METHODS AND COMPOUNDS FOR DIAGNOSING THREONYL-tRNA SYNTHETASE-ASSOCIATED DISEASES AND CONDITIONS

ABSTRACT

The invention includes, in part, methods and compounds for diagnosing diseases and conditions characterized by altered threonyl-tRNA synthetase (TARS) activity, which include, but are not limited to diseases and conditions in which angiogenesis is altered. In some embodiments of the invention, a level of a TARS molecule is determined and compared to a control level of TARS to assess onset, progression, and/or regression of a disease or condition associated with altered TARS activity.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/675,652, filed Jul. 25, 2012 and thecontent of which is incorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under RO1 GM54899 and bytraining grant T32 ES007122-23 both awarded by the National Institutesof Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates, in part, to methods and compounds for diagnosingdiseases and conditions associated with altered threonyl-tRNA synthetase(TARS) activity in a subject.

BACKGROUND

Angiogenesis plays a role in diseases such as cancer and otherproliferative disorders. For example, a small solid tumor may be able tosurvive in the absence of vascularization, but to provide sufficientnutrients and oxygen and to remove waste products from cells that makeup larger tumors, vascularization of the tissue is necessary. Triggersand regulators of angiogenesis in cells and tissues are not fullyunderstood, but it is thought that hypoxia and lack of adequatenutritional access in cells in tumors greater than approximately 2 cm³in size may result in angiogenesis, which supports further tumor growthwith increased delivery of oxygen and nutrients. Angiogenesis may be afactor in the progression of a tumor or cancer, not only by providingnutrient support for a tumor to continue to grow in size, butangiogenesis may also play a role in metastatic activity in somecancers.

Angiogenesis has emerged as a target for cancer therapy due to thereliance of many cancers on new vessels and the poor prognosisassociated with cancers that have advanced angiogenesis (Folkman, J.(2001) Semin Oncol 28 (6), 536-542). Angiogenesis is normally suppressedby angiopoietin-1 which is secreted by vascular pericytes and inhibitsendothelial cell proliferation. There are many factors involved in thetumor angiogenic switch, but initiation of angiogenesis by hypoxic tumorcells is primarily through induction of hypoxia inducible factor-1α(HIF-1α) which stimulates expression of vascular endothelial growthfactor (VEGF). VEGF acts in combination with other growth factors andreceptors to increase activation of the Ras/MAP kinase andphosphoinositide 3 kinase (PI3 kinase) pathways in endothelial cells.These pathways are involved in induction of genes involved inendothelial cell proliferation and migration. (Bikfalvi, A. andBicknell, R. (2002) Trends in Pharmacological Sciences 23 (12), 576-582;Liao, D. and Johnson, R. (2007) Cancer and Metastasis Reviews 26 (2),281-290; and Olsson, A. K. et al. (2006) Nat Rev Mol Cell Biol 7 (5),359-371).

Cell and tissue growth, for example vascular growth in angiogenesis, areknown to involve protein synthesis but processes involved in theinitiation, regulation, and modulation of protein synthesis inangiogenesis appear to be quite complex and are not well understood. Thelack of understanding of the complex pathways and interactive regulatoryevents necessary to trigger and support angiogenesis in cells limitsapproaches to diagnose disorders that are characterized, in part, byangiogenesis.

SUMMARY OF THE INVENTION

The invention includes, in part, methods and compounds for diagnosingdiseases and conditions characterized by altered threonyl-tRNAsynthetase (TARS) activity, which include, but are not limited todiseases and conditions in which angiogenesis is altered. In someembodiments of the invention, a level of a TARS molecule is determinedand compared to a control level of TARS to assess onset, progression,and/or regression of a disease or condition associated with altered TARSactivity.

It has now been shown for the first time that TARS is a potentangiogenic inducer in vitro and in vivo affecting endothelial cellmigration and tube formation. TARS is also shown to be secreted byendothelial cells in response to angiogenic or inflammatory signaling,indicating its novel role as a pro-angiogenic chemokine. Furthermore, anassociation is revealed between TARS levels and both ovarian andprostate cancers in human patient samples.

According to an aspect of the invention, methods of diagnosing a subjectas having, or at increased risk of developing a disorder associated withincreased threonyl-tRNA synthetase (TARS) activity are provided. Themethods include (a) obtaining a biological sample from a subject; (b)measuring the amount of a TARS molecule in the biological sample; and(c) comparing the level of the TARS molecule in the biological sample toa normal control level of the TARS molecule, wherein a significantincrease in the level of the TARS molecule in the subject biologicalsample compared to the normal control level is an indication that thesubject has or is at increased risk of developing the disorderassociated with increased TARS activity. In some embodiments, asignificant increase in the level of the TARS molecule in the subjectsample compared to the control determines that the subject is atincreased risk of developing a disorder associated with increased TARSactivity. In certain embodiments, the normal control is a level of theTARS molecule characteristic of biological samples from individuals freeof the disorder associated with increased TARS activity. In someembodiments, the level of the TARS molecule is measured by measuring asecreted TARS molecule. In some embodiments, the level of the TARSmolecule is measured by measuring a non-secreted TARS molecule. Incertain embodiments, the level of the TARS molecule is measured bymeasuring the activity of the TARS molecule. In some embodiments, thedisorder associated with increased TARS activity is anangiogenesis-associated disorder. In some embodiments, theangiogenesis-associated disorder is a cancer, a tumor, a hemangioma,vascular overgrowth, venous malformation, arterial malformation,overweight, macular degeneration, inflammatory disease, psoriasis,diabetes, or rheumatoid arthritis. In some embodiments, the cancer is ametastatic carcinoma of the cervix; sarcoma of the kidney; renal cellcarcinoma; prostate cancer; androgen independent prostate cancer;Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastriccancer, epithelial ovarian cancer; lung cancer, or mesothelioma. Incertain embodiments, the cancer is a metastatic cancer. In someembodiments, the biological sample is a sample of blood, tissue, serum,urine, stool, sputum, cerebrospinal fluid, or supernatant from celllysate. In some embodiments, the biological sample includes a cell ortissue. In certain embodiments, the TARS molecule includes a TARSpolypeptide. In some embodiments, the TARS molecule includes aTARS-encoding nucleic acid. In certain embodiments, a means of themeasuring includes an immunological assay, nucleic acid determination,mass spectrometry, TARS aminoacylation assay, TARS active sitedetermination assay, or a TARS binding assay including a TARS-bindingreporter molecule. In some embodiments, the immunological assay includesan ELISA assay. In some embodiments, the method also includes selectinga treatment regimen for the subject based at least in part on thecomparison of the level of the TARS molecule in the biological sample tothe normal control level of the TARS molecule. In some embodiments, thetreatment regimen includes administering one or more of a medicament,surgery, radiation, or chemotherapy to the subject. In certainembodiments, the treatment regimen includes stopping a prioradministration regimen of one or more of a medicament, surgery,radiation, or chemotherapy to the subject. In some embodiments, thecontrol is a sample obtained from the subject at a different time thanthe sample obtained in step (a). In some embodiments, the method alsoincludes administering one or more of a medicament, surgery, radiation,or chemotherapy to the subject to treat the disorder, wherein theselection of the one of more of the medicament, surgery, radiation, orchemotherapy is selected based, at least in part, on the diagnosis.

According to another aspect of the invention, methods of diagnosing asubject as having, or at increased risk of developing, a disorderassociated with increased threonyl-tRNA synthetase (TARS) activity areprovided. The methods include (a) obtaining a biological sample from asubject; (b) measuring the amount of a TARS molecule in the biologicalsample; and (c) comparing the level of the TARS molecule in thebiological sample to a disease control level of the TARS molecule,wherein a similar level of the TARS molecule in the subject biologicalsample compared to the disease control level is an indication that thesubject has or is at increased risk of developing the disorderassociated with increased TARS activity. In certain embodiments, thedisease control level is a level of the TARS molecule characteristic ofbiological samples from individuals having the disorder associated withincreased TARS activity. In some embodiments, the level of the TARSmolecule is measured by measuring a secreted TARS molecule. In someembodiments, the level of the TARS molecule is measured by measuring anon-secreted TARS molecule. In some embodiments, the level of the TARSmolecule is measured by measuring the activity of TARS. In certainembodiments, the disorder associated with increased TARS activity is anangiogenesis-associated disorder. In some embodiments, theangiogenesis-associated disorder is a cancer, a tumor, a hemangioma,vascular overgrowth, venous malformation, arterial malformation,overweight, macular degeneration, inflammatory disease, psoriasis,diabetes, or rheumatoid arthritis. In some embodiments, the cancer is ametastatic carcinoma of the cervix; sarcoma of the kidney; renal cellcarcinoma; prostate cancer; androgen independent prostate cancer;Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastriccancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In someembodiments, the cancer is a metastatic cancer. In certain embodiments,the biological sample is a sample of blood, tissue, serum, urine, stool,sputum, cerebrospinal fluid, or supernatant from cell lysate. In someembodiments, the biological sample includes a cell or tissue. In someembodiments, the TARS molecule is a TARS polypeptide. In someembodiments, the TARS molecule is a TARS-encoding nucleic acid. Incertain embodiments, a means for the measuring includes an immunologicalassay, nucleotide determination (mRNA or DNA), mass spectrometry, TARSaminoacylation assay, TARS active site determination assay, a TARSbinding assay including a TARS-binding reporter molecule, or an ELISAassay. In some embodiments, the method also includes selecting atreatment regimen for the subject based at least in part on thecomparison of the level of the TARS molecule in the biological sample tothe normal control level of the TARS molecule. In some embodiments, thetreatment regimen includes administering one or more of a medicament,surgery, radiation, or chemotherapy to the subject. In certainembodiments, the treatment regimen includes stopping a prioradministration regimen of one or more of a medicament, surgery,radiation, or chemotherapy to the subject. In some embodiments, thecontrol is a sample obtained from the subject at a different time thanthe sample obtained in step (a). In some embodiments, the method alsoincludes administering one or more of a medicament, surgery, radiation,or chemotherapy to the subject to treat the disorder, wherein theselection of the one of more of the medicament, surgery, radiation, orchemotherapy is selected based, at least in part, on the diagnosis.

According to another aspect of the invention, methods for determining aprognosis in a subject diagnosed with a disorder associated withincreased threonyl-tRNA synthetase (TARS) activity are provided. Themethods include (a) obtaining a biological sample from the subject; (b)measuring a TARS molecule level in the subject's biological sample; (c)comparing the TARS molecule level of the subject's sample with a controllevel of the TARS molecule; and (d) correlating levels of the TARSmolecule greater than the control level with an indication ofunfavorable prognosis and levels of the TARS molecule at or below thecontrol level with a favorable prognosis. In certain embodiments, thedisorder associated with increased TARS activity is anangiogenesis-associated disorder. In some embodiments, theangiogenesis-associated disorder is a cancer, a tumor, a hemangioma,vascular overgrowth, venous malformation, arterial malformation,overweight, macular degeneration, inflammatory disease, psoriasis,diabetes, or rheumatoid arthritis. In some embodiments, the cancer is ametastatic carcinoma of the cervix; sarcoma of the kidney; renal cellcarcinoma; prostate cancer; androgen independent prostate cancer;Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastriccancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In someembodiments, the cancer is a metastatic cancer. In certain embodiments,the TARS molecule is a TARS protein. In some embodiments, the TARSmolecule is a TARS-encoding nucleic acid. In some embodiments, a meansof the measuring includes an immunological assay, nucleotidedetermination (mRNA or DNA), mass spectrometry, TARS aminoacylationassay, TARS active site determination assay, or a TARS binding assayincluding a TARS-binding reporter molecule, or an ELISA assay. Incertain embodiments, the method also includes selecting a treatmentregimen for the subject based at least in part on the determinedprognosis. In some embodiments, the treatment regimen includesadministering one or more of a medicament, surgery, radiation, orchemotherapy to the subject. In certain embodiments, the treatmentregimen includes stopping a prior administration regimen of one or moreof a medicament, surgery, radiation, or chemotherapy to the subject. Insome embodiments, the control is a sample obtained from the subject at adifferent time than the sample obtained in step (a). In someembodiments, the method also includes administering one or more of amedicament, surgery, radiation, or chemotherapy to the subject to treatthe disorder, wherein the selection of the one of more of themedicament, surgery, radiation, or chemotherapy is selected based, atleast in part, on the prognosis determined.

According to another aspect of the invention, methods of determining theonset, progression, or regression in a subject of a disorder associatedwith increased threonyl-tRNA synthetase (TARS) activity are provided.The methods include (a) obtaining a first biological sample from asubject; (b) measuring the level of a TARS molecule in the firstbiological sample; (c) obtaining a second biological sample from thesubject; (d) measuring the amount of the TARS molecule in the secondbiological sample obtained from the subject, wherein the secondbiological sample is obtained from the subject at a time subsequent tothe time the first biological sample is obtained; and (e) comparing themeasurement of the TARS molecule in the first biological sample to themeasurement of the TARS molecule in the second biological sample as adetermination of the onset, progression, or regression of the disorderassociated with increased TARS activity. In some embodiments, anincrease in the level of the TARS molecule in the second sample comparedto the first sample indicates the onset or progression of the disorder.In certain embodiments, a decrease in the level of the TARS molecule inthe second sample compared to the first sample indicates the regressionof the disorder. In some embodiments, the disorder associated withincreased TARS activity is an angiogenesis-associated disorder. In someembodiments, the angiogenesis-associated disorder is a cancer, a tumor,a hemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, or rheumatoid arthritis. In certain embodiments,the cancer is a metastatic carcinoma of the cervix; sarcoma of thekidney; renal cell carcinoma; prostate cancer; androgen independentprostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliarycancer, gastric cancer, epithelial ovarian cancer; lung cancer, ormesothelioma. In some embodiments, the cancer is a metastatic cancer. Insome embodiments, the TARS molecule is a TARS protein. In someembodiments, the TARS molecule is a TARS-encoding nucleic acid. Incertain embodiments, a means of the measuring includes an immunologicalassay, nucleotide determination (mRNA or DNA), mass spectrometry, TARSaminoacylation assay, TARS active site determination assay, a TARSbinding assay including a TARS-binding reporter molecule, or an ELISAassay. In some embodiments, the method also includes selecting atreatment regimen for the subject based at least in part on thedetermination of the onset, progression or regression of the disorder.In some embodiments, the treatment regimen includes administering one ormore of a medicament, surgery, radiation, or chemotherapy to thesubject. In certain embodiments, the treatment regimen includes stoppinga prior administration of one or more of a medicament, surgery,radiation, or chemotherapy. In some embodiments, the method alsoincludes administering one or more of a medicament, surgery, radiation,or chemotherapy to the subject to treat the disorder, wherein theselection of the one of more of the medicament, surgery, radiation, orchemotherapy is selected based, at least in part, on the determinedonset, progression or regression of the of the disorder.

According to yet another aspect of the invention, methods fordetermining the metastatic potential of a cancer are provided. Themethods include (a) obtaining a biological sample from a subject havinga cancer; (b) measuring a level of a threonyl-tRNA synthetase (TARS)molecule in the biological sample; and (c) comparing the level of theTARS molecule in the biological sample to a control level of the TARSmolecule, wherein a significantly higher level of the TARS molecule inthe sample compared to the control level of the TARS molecule indicatesa higher metastatic potential for the cancer. In some embodiments, thecancer is a carcinoma of the cervix; sarcoma of the kidney; renal cellcarcinoma; prostate cancer; androgen independent prostate cancer;Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastriccancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In someembodiments, the TARS molecule is a TARS protein. In certainembodiments, the TARS molecule is a TARS-encoding nucleic acid. In someembodiments, a means for the measuring includes an immunological assay,nucleotide determination (mRNA or DNA), mass spectrometry, TARSaminoacylation, GTPase, or Ap4A synthesis assay, TARS active sitedetermination assay, a TARS binding assay including a TARS-bindingreporter molecule, or an ELISA assay. In some embodiments, the methodalso includes selecting a treatment regimen for the subject based atleast in part on the determined metastatic potential. In certainembodiments, the treatment regimen includes administering one or more ofa medicament, surgery, radiation, or chemotherapy to the subject. Insome embodiments, the treatment regimen includes stopping a prioradministration regimen of one or more of a medicament, surgery,radiation, or chemotherapy to the subject. In some embodiments, thecontrol is a sample obtained from the subject at a different time thanthe sample obtained in step (a). In some embodiments, the method alsoincludes administering one or more of a medicament, surgery, radiation,or chemotherapy to the subject to treat the disorder, wherein theselection of the one of more of the medicament, surgery, radiation, orchemotherapy is selected based, at least in part, on the determinedmetastatic potential.

According to another aspect of the invention, methods of diagnosing asubject as having, or is at increased risk of developing, a disorderassociated with decreased threonyl-tRNA synthetase (TARS) activity areprovided. The methods include (a) obtaining a biological sample from asubject; (b) measuring the amount of a TARS molecule in the biologicalsample; (c) comparing the level of the TARS molecule in the biologicalsample to a normal control level of the TARS molecule, wherein asignificant decrease in the level of the TARS molecule in the subjectbiological sample compared to the normal control level is an indicationthat the subject has or is at increased risk of developing the disorderassociated with decreased TARS activity. In certain embodiments, asignificant decrease in the level of the TARS molecule in the subjectsample compared to the control determines that the subject is at risk ofdeveloping a disorder associated with decreased TARS activity. In someembodiments, the normal control is a level of the TARS moleculecharacteristic of biological samples from individuals free of thedisorder associated with decreased TARS activity. In some embodiments,the level of the TARS molecule is measured by measuring a secreted TARSmolecule. In certain embodiments, the level of the TARS molecule ismeasured by measuring a non-secreted TARS molecule. In some embodiments,the level of the TARS molecule is measured by measuring the activity ofthe TARS molecule. In some embodiments, the disorder associated withdecreased TARS activity is an angiogenesis-associated disorder. In someembodiments, the subject has or is at risk of having anangiogenesis-associated disease or condition. In certain embodiments,the angiogenesis-associated disease or condition is a tissue implant,organ implant, ischemia, peripheral vascular disease, diabetes, cardiacinfarction, tissue trauma, cartilage to bone transformation, stroke,surgery, pregnancy, macular degeneration, or vascular occlusion. In someembodiments, the biological sample is a sample of blood, tissue, serum,urine, stool, sputum, cerebrospinal fluid, or supernatant from celllysate. In some embodiments, the biological sample includes a cell ortissue. In certain embodiments, the TARS molecule includes a TARSpolypeptide. In some embodiments, the TARS molecule includes aTARS-encoding nucleic acid. In some embodiments, a means for themeasuring includes an immunological assay, nucleotide determination(mRNA or DNA), mass spectrometry, TARS aminoacylation assay, TARS activesite determination assay, a TARS binding assay including a TARS-bindingreporter molecule, or an ELISA assay. In some embodiments, the methodalso includes selecting a treatment regimen for the subject based atleast in part on the determined diagnosis. In certain embodiments, thetreatment regimen includes administering one or more of a medicament,surgery, radiation, or chemotherapy to the subject. In some embodiments,the treatment regimen includes stopping a prior administration regimenof one or more of a medicament, surgery, radiation, or chemotherapy tothe subject. In some embodiments, the control is a sample obtained fromthe subject at a different time than the sample obtained in step (a). Insome embodiments, the method also includes administering one or more ofa medicament, surgery, radiation, or chemotherapy to the subject totreat the disorder, wherein the selection of the one of more of themedicament, surgery, radiation, or chemotherapy is selected based, atleast in part, on the diagnosis determined.

According to yet another aspect of the invention, methods of diagnosinga subject as having, or at increased risk of developing, a disorderassociated with decreased threonyl-tRNA synthetase (TARS) activity areprovided. The methods include (a) obtaining a biological sample from thesubject; (b) measuring the amount of a TARS molecule in the biologicalsample; and (c) comparing the level of the TARS molecule in thebiological sample to a disease control level of the TARS molecule,wherein a similar level of the TARS molecule in the subject biologicalsample compared to the disease control level is diagnostic for thesubject having or being at increased risk of developing the disorderassociated with decreased TARS activity. In certain embodiments, thedisease control level is a level of the TARS molecule characteristic ofbiological samples from individuals having the disorder associated withdecreased TARS activity. In some embodiments, the level of the TARSmolecule is measured by measuring the level of a secreted TARS molecule.In some embodiments, the level of the TARS molecule is measured bymeasuring a non-secreted TARS molecule. In certain embodiments, thelevel of the TARS molecule is measured by measuring the activity ofTARS. In some embodiments, the disorder associated with decreased TARSactivity is an angiogenesis-associated disorder. In some embodiments,the angiogenesis-associated disease or condition is a tissue implant,organ implant, ischemia, cardiac infarction, tissue trauma, cartilage tobone transformation, stroke, peripheral vascular disease, surgery,pregnancy, macular degeneration, or vascular occlusion. In someembodiments, the biological sample is a sample of blood, tissue, serum,urine, stool, sputum, cerebrospinal fluid, or supernatant from celllysate. In certain embodiments, the biological sample includes a cell ortissue. In some embodiments, the TARS molecule is a TARS protein. Insome embodiments, the TARS molecule is a TARS-encoding nucleic acid. Insome embodiments, a means for the measuring includes an immunologicalassay, nucleotide determination (mRNA or DNA), mass spectrometry, TARSaminoacylation assay, TARS active site determination assay, a TARSbinding assay including a TARS-binding reporter molecule, or an ELISAassay. In certain embodiments, the method also includes selecting atreatment regimen for the subject based at least in part on thedetermined diagnosis. In some embodiments, the treatment regimenincludes administering one or more of a medicament, surgery, radiation,or chemotherapy to the subject. In some embodiments, the treatmentregimen includes stopping a prior administration regimen of one or moreof a medicament, surgery, radiation, or chemotherapy to the subject. Incertain embodiments, the control is a sample obtained from the subjectat a different time than the sample obtained in step (a). In someembodiments, the method also includes administering one or more of amedicament, surgery, radiation, or chemotherapy to the subject to treatthe disorder, wherein the selection of the one of more of themedicament, surgery, radiation, or chemotherapy is selected based, atleast in part, on the diagnosis determined.

According to yet another aspect of the invention, methods fordetermining a prognosis in a subject diagnosed with a disorderassociated with decreased threonyl-tRNA synthetase (TARS) activity areprovided. The methods include (a) obtaining a biological sample from thesubject; (b) measuring a TARS molecule level in the subject's biologicalsample; (c) comparing the TARS molecule level of the subject's samplewith a control level of the TARS molecule; and (d) correlating levels ofthe TARS molecule lower than the control level with an indication ofunfavorable prognosis and levels of the TARS molecule at or above thecontrol level with a favorable prognosis. In some embodiments, thedisorder associated with decreased TARS activity is anangiogenesis-associated disorder. In certain embodiments, theangiogenesis-associated disease or condition is a tissue implant, organimplant, ischemia, cardiac infarction, tissue trauma, cartilage to bonetransformation, stroke, peripheral vascular disease, diabetes, surgery,pregnancy, macular degeneration, vascular occlusion. In someembodiments, the TARS molecule is a TARS protein. In some embodiments,the TARS molecule is a TARS-encoding nucleic acid. In some embodiments,a means for the measuring includes an immunological assay, nucleotidedetermination (mRNA or DNA), mass spectrometry, TARS aminoacylationassay, TARS active site determination assay, a TARS binding assayincluding a TARS-binding reporter molecule, or an ELISA assay. Incertain embodiments, the method also includes selecting a treatmentregimen for the subject based at least in part on the determinedprognosis. In some embodiments, the treatment regimen includesadministering one or more of a medicament, surgery, radiation, orchemotherapy to the subject. In some embodiments, the treatment regimenincludes stopping a prior administration regimen of one or more of amedicament, surgery, radiation, or chemotherapy to the subject. In someembodiments, the control is a sample obtained from the subject at adifferent time than the sample obtained in step (a). In certainembodiments, the method also includes administering one or more of amedicament, surgery, radiation, or chemotherapy to the subject to treatthe disorder, wherein the selection of the one of more of themedicament, surgery, radiation, or chemotherapy is selected based, atleast in part, on the prognosis determined.

According to another aspect of the invention, methods of determining theonset, progression, or regression in a subject of a disorder associatedwith decreased threonyl-tRNA synthetase (TARS) activity are provided.The methods include (a) measuring the level of a TARS molecule in afirst biological sample obtained from a subject, (b) measuring theamount of the TARS molecule in a second biological sample obtained fromthe subject, wherein the second biological sample is obtained from thesubject at a time subsequent to the time the first biological sample isobtained; and (c) comparing the measurement of the TARS molecule in thefirst sample to the measurement of the TARS molecule in the secondsample as a determination of the onset, progression, or regression ofthe disorder associated with decreased TARS activity. In someembodiments, a decrease in the level of the TARS molecule in the secondsample compared to the first sample indicates the onset or progressionof the disorder. In some embodiments, an increase in the level of theTARS molecule in the second sample compared to the first sampleindicates the regression of the disorder. In some embodiments, thedisorder associated with decreased TARS activity is anangiogenesis-associated disorder. In certain embodiments, theangiogenesis-associated disease or condition is a tissue implant, organimplant, ischemia, cardiac infarction, tissue trauma, cartilage to bonetransformation, stroke, surgery, peripheral vascular disease, diabetes,pregnancy, macular degeneration, or vascular occlusion. In someembodiments, the TARS molecule is a TARS protein. In some embodiments,the TARS molecule is a TARS-encoding nucleic acid. In some embodiments,a means for the measuring includes an immunological assay, nucleotidedetermination (mRNA or DNA), mass spectrometry, TARS aminoacylationassay, TARS active site determination assay, a TARS binding assayincluding a TARS-binding reporter molecule, or an ELISA assay. Incertain embodiments, the method also includes selecting a treatmentregimen for the subject based at least in part on the determination ofthe onset, progression or regression of the disorder. In someembodiments, the treatment regimen includes administering one or more ofa medicament, surgery, radiation, or chemotherapy to the subject. Insome embodiments, the treatment regimen includes stopping a prioradministration of one or more of a medicament, surgery, radiation, orchemotherapy. In certain embodiments, the method also includesadministering one or more of a medicament, surgery, radiation, orchemotherapy to the subject to treat the disorder, wherein the selectionof the one of more of the medicament, surgery, radiation, orchemotherapy is selected based, at least in part, on the determinedonset, progression or regression of the of the disorder.

According to another aspect of the invention, kits are provided. Thekits including a means for measuring activity of a threonyl-tRNAsynthase (TARS) molecule and instructions for measuring a level of TARSactivity in a biological sample obtained from a subject. In someembodiments, the kit also includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore compounds that bind to or react with a TARS molecule, and a meansfor detecting the binding or reaction. In some embodiments, the kit alsoincludes one or more detectable labels and instructions for using theone or more detectable labels to measure TARS activity in a biologicalsample. In some embodiments, the TARS molecule is a TARS protein. Incertain embodiments, the TARS molecule is a TARS-encoding nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D provides photomicrographic images and a graph providingevidence that a subnanomolar concentration of the TARS inhibitor BC194inhibits endothelial tube formation. Human umbilical vein endothelialcells (HUVECs) were seeded on Matrigel™ in full serum media (2% FBS)along with the indicated concentrations of BC194. After 6 h, cells werefixed and stained with Oregon Green 488 Phalloidin. Shown arerepresentative images using the full serum media response as Control(FIG. 1A); FIG. 1B shows results from 10 nM BC194, and FIG. 1C showsresults from 1000 nM BC194. Scale bar=100 μm. Graph in FIG. 1D showsquantification of branches over a range of BC194 concentrations usingthe Simple Neurite Tracer plug-in on ImageJ software. Numbers representaverage data from 3 separate experiments performed in duplicate.Multiple comparisons of one-way ANOVA were performed using the TukeyTest; n=3, *p<0.05.

FIG. 2 is a graph showing that BC194 does not affect endothelial tubebranch length. HUVECS were treated with the indicated concentration ofthe TARS inhibitor BC194. Graph shows quantification of branch length inpixels over a range of BC194 concentrations using the Simple NeuriteTracer plug-in on ImageJ software. Multiple comparisons of one-way ANOVAwere performed using the Tukey Test; n=3.

FIG. 3A-D shows Western blots and graphs that indicate that highconcentrations of BC194 are required to stimulate the unfolded proteinresponse and apoptosis. HUVECs grown in full serum media were exposed tothe indicated concentrations of BC194, followed by Western Blot of cellextracts using antibodies recognizing phospho-eIF2α (FIG. 3A), cleavedcaspase-3 (FIG. 3B) and β-actin or β-tubulin as a loading control.Quantification of phospho-eIF2α and c-caspase-3 relative to the loadingcontrols were determined using Quantity One software and average dataare shown in FIGS. 3C and 3D; respectively *p<0.05, n=3.

FIG. 4A-D provides graphs and an SDS-PAGE illustrating a lack of effectof BC194 on cell viability and protein synthesis. FIG. 4A is a graphshowing effects of BC194 on cell viability. HUVECs were exposed to theindicated concentrations of BC194, and live cells were quantified bytrypan blue exclusion and normalized to the untreated control; n=3,*p<0.05. FIG. 4B is a graph showing effects of BC194 on proliferation.HUVECs were exposed to the indicated concentrations of BC194 andproliferation was quantified over time using an Alamar Blue™ assay (ameasure of NADPH oxidase activity). n=3, *p<0.05. FIG. 4C is a flowcytometry analysis, and FIG. 4D is an SDS-PAGE showing lack of effectsof BC194 on nascent protein synthesis. Cells were exposed to theindicated concentrations of BC194 and new protein synthesis was detectedusing a Click-iT® metabolic labeling kit. Proteins were separated bySDS-PAGE and visualized using streptavidin-HRP. Cycloheximide (CHX, 50μM) was used as a control for complete inhibition of protein synthesis.

FIG. 5A-B provides a blot and graphs indicating expression and activityof human TARS and L567V TARS. Proteins were expressed and purified fromE. coli Rosetta™ cells as described in Examples section. FIG. 5A showsresults using Coomassie stain of TARS and L567V TARS proteins separatedby SDS-PAGE indicating purified intact proteins. FIG. 5B is a graphshowing that purified TARS exhibits aminoacyl synthetase activity andactivity is not compromised in the borrelidin-resistant mutant L567V.TARS activity was comparable to E. coli TARS and commercially availablehuman TARS expressed in CHO cells (Francklyn, First et al. 2008).

FIG. 6A-F provides photomicrographic images and a graph providingevidence that exogenous application of TARS promotes angiogenesis by anin vitro endothelial tube formation assay. FIGS. 6A, 6B, and 6C showresults using low serum, full serum, and low serum+TARS, respectively.HUVECs were plated onto Matrigel in low serum (LS, 0.2% fetal bovineserum) or EGM-2 full serum media (FS, 5% FBS). Where indicated, 100 nMpurified recombinant human TARS was added to the media. Tubes wereimaged and analyzed after 6 h as in FIG. 1, Scale bar=100 μm. FIG. 6D isa histograph of quantified branches; n=3, *p<0.01 compared to low serum.FIG. 6E is a histogram of quantified branches for TARS effect;mean±standard error of the mean, n=3, *P,0.01 compared to Low Serum(Student's test). FIG. 6F is a histogram of quantified branches for arange of BC194 concentrations added to Full Serum media. Numbersrepresent mean±standard error of the mean, n=3, *P<0.05 (one-way ANOVA,Tukey test).

FIG. 7A-C provides graphs and a photomicrographic image demonstratingthat TARS induces in vivo angiogenesis in a CAM assay. Fertilizedchicken embryos were cultured ex-ova and, starting at developmental day10, agents were applied daily to gelfoam sponges on the CAM. Images wererecorded daily over 72 h and scored blindly according to a modifiedversion of Intensity Scoring as previously described (Ribatti et al,2006). Graphs represent the change in CAM vascularity score over 72 h.(FIG. 7A) BC194 (10 nM) was applied to the CAM along with PBS (Control),bFGF (40 μg/ml), and VEGF (2 μg/ml). The angiostatic control retinoicacid (RA) was used at 100 μg/ml. Representative images for (FIG. 7A) areshown in FIG. 8B, and FIG. 8C. Purified recombinant TARS,BC194-resistant mutant TARS (L567V) and BC194 were applied at 100 μg/ml.FIG. 7C shows representative CAM images over time; arrows denotespoke-wheel response. Scale bar=1.0 mm. FIG. 7B is a histogram of changein CAM vascularity score over 72 h; n>14, *p<0.001 compared to PBScontrol; #p<0.001 compared to TARS.

FIG. 8A-N provides representative photomicrographic images for thegraphs shown in FIGS. 7A and 7B. Fertilized chicken embryos werecultured ex-ova and, starting at developmental day 10, agents wereapplied daily to gelfoam sponges on the CAM. Left panels representimages taken at 24 h and right panels at 72 h for PBS Control (FIGS. 8Aand 8B), bFGF (FIGS. 8C-F), VEGF (FIGS. 8G-J) and L567V TARS (FIGS.8K-N). BC194 (100 ng/sponge) was included where indicated. Arrowsindicate spoke-wheel response; Scale bar=1.0 mm. FIG. 9A-D provides ablot, graph, photomicrographic image, and a histogram showing thepurification, activity, and lack of CAM effects of Leucyl tRNAsynthetase (LARS). Human LARS was purified from E. coli as described inMaterials and Methods in Examples section FIG. 9A shows Coomassie stainof 2 μg LARS separated by SDS-PAGE. FIG. 9B shows a graph indicatingthat LARS exhibits enzyme activity as measured by conversion of ³²P-ATPto AMP. Numbers represent labeled AMP determined by thin layerchromatography followed by phosphorimaging. FIGS. 9C and D indicate thatLARS has no effect on angiogenesis measured in the CAM assay. PurifiedLARS (100 ng/sponge) was added to CAMs as in FIG. 8. FIG. 9C providesrepresentative images showing no effect of LARS on CAM vascularity;Scale bar=1.0 mm. FIG. 9D provides a graph representing the average CAMvascularity score over 72 h as compared to PBS or TARS; n=5, *p<0.05.

FIG. 10A-D provides graphs and a Western blot demonstrating that TARS issecreted by endothelial cells in response to VEGF and TNF-α. In FIG. 10AHUVECs were treated with VEGF or TNF-α (50 ng/ml) where indicated. After6 h the level of TARS in the supernatant was determined by ELISA. Graphrepresents an average of 3 experiments; *p<0.05. FIG. 10B shows cellmembrane integrity for the experiments in (A and C) using the lactatedehydrogenase assay CytoTox-ONE™ at 6 h and 16 h. Numbers representpercent cytotoxicity relative to a lysis control. For FIG. 10C HUVECsgrown on a 10 cm dish were exposed to 50 ng/ml of VEGF or TNF-α in 0%serum EGM-2 media for 16 h. Shown is a representative TARS Western blotof cell lysates and media samples, n=4. Media was concentrated 20-foldto accommodate 25% onto the gel and compared to 5% of the cell lysate.Purified TARS was used to estimate the TARS concentration withinsamples. β-tubulin was measured as a loading and lysis control. FIG. 10Dshows that VEGF and TNF-α do not induce TARS transcription. HUVECs wereexposed to 50 ng/ml of VEGF or TNF-α followed by RNA extraction andRT-qPCR to measure TARS mRNA levels. Shown are Rq values relative to aβ-2 macroglobulin control; n=3. FIG. 10E shows that TARS does not induceVEGF secretion. HUVECs were exposed to the indicated concentrations ofpurified recombinant human TARS for 24 h and the level of VEGF in thesupernatant determined by ELISA; n=3.

FIG. 11A-C provides graphs, photomicrographic images and a histogramshowing that TARS selectively induces migration of endothelial cellsthat is sensitive to BC194. FIG. 11A shows results indicating that TARSdoes not significantly affect cell proliferation. HUVECs were culturedin low serum (0.2% FBS) media containing 50 ng/ml VEGF and 10 nM BC194where indicated; relative proliferation was measured over time using anAlamar Blue™ assay; n=3. FIGS. 11B and C show VEGF and TARS-mediatedmigration. HUVEC migration was measured using a trans-well assay. Themigration compartment contained 50 ng/ml VEGF, 100 nM LARS or 100 nMTARS and 10 nM BC194 where indicated. Shown in FIG. 11B arerepresentative images of DAPI stained nuclei from migrated cells after 4h. FIG. 11C shows a histogram representing number of migrated cellsafter 4 h for the conditions indicated; n>3, *p<0.05 compared toControl, #p<0.05 compared to VEGF.

FIG. 12 is a schematic diagram of a proposed model for TARS signalingand angiogenic activity. VEGF and TNF-αt secretion by hypoxic and cellsof the tumor microenvironment leads to VEGF receptor activation onendothelial cells and secretion of TARS. Secreted TARS has autocrine andpossibly paracrine functions that promote angiogenic signaling. BC194binds and inactivates TARS, preventing its angiogenic function. Thus,TARS present in patient serum could be an indicator of the angiogenicpotential of tumors.

FIG. 13A-C provides photomicrographs and a histogram of statisticalanalysis correlating TARS levels in prostate cancer tissue sections withGleason score, and a table depicting the results of initial ELISAmeasurements on serum samples from prostate cancer patients. The imagesin FIG. 13A show results of immunohistochemistry of TARS within patienttissue sections showing examples of the scoring rubric. (i=TARS+1,ii=TARS+2, iii=TARS+3, iv=Atrophy, v=benign prostate hyperplasia (BPH)TARS+1, vi=BPH TARS negative, and vii=TARS negative). FIG. 13B presentsa graph representing statistical analysis of TARS expression score asrelated to tumor diagnosis. Slides were scored by at least twopathologists, with a third tie breaker when necessary. Values withinbars represent number of patients. *p<0.0001. FIG. 13C presents a tabledescribing TARS serum measurements in four age matched control subjectsand ten prostate cancer patients in various stages of diagnosis andtreatment.

FIG. 14 shows a Western blot demonstrating that TARS inhibitors reduceHIF-1α stabilization in hypoxia. SKOV-3 cells were exposed to hypoxia(2% 02) for 6 h in the presence of the indicated concentrations of theTARS inhibitors: borrelidin (BC144) or BC194. CoCl₂ was used as apositive control for HIF-1α stabilization. HIF-1α and TARS proteins weredetected by Western blot.

FIG. 15A-B provides evidence for an interaction between TARS and the vonHippel Lindau protein (VHL). Plasmids expressing biotinylatable TARS(TARS-HA-Biotin) and myc-tagged VHL (VHL-myc) were transfected intoHEK293 cells, and then extracts were prepared. Biotin-TARS wasprecipitated using streptavidin-coupled beads. Myc-VHL was precipitatedusing anti-myc antibodies. Shown in FIG. 15A is an interaction betweenfull-length TARS and VHL by co-immunoprecipitation. Top panels are blotsprobed with antibody against HA (anti-TARS), bottom panels are blotsprobed with antibody against myc (VHL) antibody. The various lanesindicate input lysates (Input), streptavidin affinity purified(AP:Biotin), anti-Myc immunoprecipitates (IP:Myc), naïve IgGimmunoprecipitates IIP:IgG). Shown in FIG. 15B is the same experiment asFIG. 15A, only the N1 domain of TARS is used in place of the full lengthenzyme. See FIG. 16 for structural details.

FIG. 16 lists the putative interacting partners of TARS, as determinedfrom an affinity purification-mass spectrometry experiment. In allexperiments, TARS was over-expressed (with a biotinylatable tag tail)and then affinity purified on streptavidin-conjugated beads. The boundproteins were then removed from the beads by boiling, resolved on SDSpolyacrylamide gels, and then extracted from individual gel slices. Theexperiment was performed under two conditions. In Condition 1, only TARSwas overexpressed; in Condition 2, both TARS and VHL were overexpressed.

FIG. 17A-F provides graphs and histogram showing that human TARS doesnot require aminoacylation activity to stimulate angiogenesis activity,and is capable of catalyzing nucleotidase and nucleotide synthesisreactions distinct from aminoacylation. Wild type and R442A mutant TARSwere produced as described in Materials and Methods, in Examplessection. The R442A mutant substitution exchanges an essential catalyticarginine in the TARS active site for an alanine. The CAM assays wereperformed as described in Material and Methods in Examples section. FIG.17A is a graph comparing the aminoacylation progress curves of wild andR442A mutant TARS. Based on the results of this assay, R442A TARS hasvirtually negligible aminoacylation function. FIG. 17B compares thechange in CAM vascularity score for wild type, BC-194 resistant L567VTARS and aminoacylation-deficient R442A TARS. The histograms representthe change in vascularity score over 72 hour; *p<0.001 compared to PBScontrol; #P<0.001 compared to TARS. FIG. 17C is a graph comparing theprogress curves of Ap4A formation for human TARS, R442A TARS, and TARSin the presence of BC194 or borrelidin. FIG. 17D is a graph comparingthe progress curves of Ap4A formation for human TARS, R442A TARS, andTARS in the presence of (10 μM) BC194 or borrelidin (10 μM). FIGS. 17Cand 17D indicate that Ap4A and Ap4G synthesis is blocked in R442A TARS,and its synthesis is at least partially inhibited by borrelidin andBC914. FIG. 17E is a graph comparing the progress curves of GTPhydrolysis for human TARS, R442A TARS, and E. coli ThrRS in the presenceof (10 mM) BC194 or borrelidin. This plot indicates that wild type humanand R442 TARS both possess potent GTPase activities, but the bacterialenzyme does not. FIG. 17F is a graph comparing the progress curves ofGTP hydrolysis for human TARS in the presence of substrates that arespecific for the aminoacylation reaction. The key result is that whenATP and aminoacylation substrates are present, GTPase function isseverely inhibited.

FIG. 18A-F provides photomicrograph images and graphs showing TARSexpression by immunohistochemistry (IHC) is increased in human serouspapilloma ovarian cancer and colocalizes with VEGF. Patient tumorsamples were sectioned, and stained using anti-TARS (FIG. 18B) oranti-VEGF (FIG. 18C) antibodies. Control (FIG. 18A) for staining had noprimary antibody (No Ab). Slides were lightly stained with hematoxylinand eosin for visualizing cell structures. Statistical analysis showsexpression of TARS is significantly increased in ovarian cancer (FIG.18D), and regression analysis correlates TARS in tumor tissue withlevels of VEGF (FIG. 18E) and serum levels of TARS (FIG. 18F) asmeasured by ELISA.

Amino Acid and Nucleotide SequencesSEQ ID NO: 1 Human TARS nucleic acid sequence is provided as GENBANK ™Accession No. NM_152295 mRNA.ggtcagcggagagtaggcatgtagcttctgcagttgctcctcctcaccctccgcgacctgatttcctagaagggctctgtcacccgaaaagattttccactggcttagaggagggagggcccgccttcccccgttatccattggctgctcgttccgccgcaagttgggggcggggttagggcgcctttcgattgcatcagctggtccagccgaggccaagtcccgggcgctagcccacctcccacccgcctcttggctcctctcctctaggccgtcgctttcgggttctctcatcgcttcgtcgttcgccaatgtttgaggagaaggccagcagtccttcagggaagatgggaggcgaggagaagccgattggtgctggtgaagagaagcaaaaggaaggaggcaaaaagaagaacaaagaaggatctggagatggaggtcgagctgagttgaatccttggcctgaatatatttacacacgtcttgagatgtataatatactaaaagcagaacatgattccattctggcagaaaaggcagaaaaagatagcaagccaattaaagtcactttgcctgatggtaaacaggttgatgcggaatcttggaaaactacaccatatcaaattgcctgtggaattagtcaaggcctggccgacaacaccgttattgctaaagtaaataatgttgtgtgggacctggaccgccctctggaagaagattgtaccttggagcttctcaagtttgaggatgaggaagctcaggcagtgtattggcactctagtgctcacataatgggtgaagccatggaaagagtctatggtggatgtttatgctacggtccgccaatagaaaatggattctattatgacatgtacctcgaagaagggggtgtgtctagcaatgatttctcttctctggaggctttgtgtaagaaaatcattaaagaaaaacaagcttttgaaagactggaagttaagaaagaaactttactggcaatgtttaagtacaacaagttcaaatgccggatattgaatgaaaaggtgaatactccaactaccacagtctatagatgtggccctttgatagatctctgccggggtcctcatgttagacacacgggcaaaattaaggctttaaaaatacacaaaaattcctccacgtactgggaaggcaaagcagatatggagactctccagagaatttatggcatttcattcccagatcctaaaatgttgaaagagtgggagaagttccaagaggaagctaaaaaccgagatcataggaaaattggcagggaccaagaactatatttctttcatgaactcagccctggaagttgcattactgccaaaaggagcctacatttataatgcacttattgaattcattaggagcgaatataggaaaagaggattccaggaggtagtcaccccaaacatcttcaacagccgactctggatgacctcgggccactggcagcactacagcgagaacatgttctcctttgaggtggagaaggagctgtttgccctgaaacccatgaactgcccaggacactgccttatgtttgatcatcggccaaggtcctggcgagaactgcctctgcggctagctgattttggggtacttcataggaacgagctgtctggagcactcacaggactcacccgggtacgaagattccaacaggatgatgctcacatattctgtgccatggagcagattgaagatgaaataaaaggttgtttggattttctacgtacggtatatagcgtatttggattttcttttaaactaaacctttctactcgcccggaaaaattccttggagatatcgaagtatgggatcaagctgagaaacaacttgaaaacagtctgaatgaatttggtgaaaagtgggagttaaactctggagatggagctttctatggcccaaagattgacatacagattaaagatgcgattgggcggtaccaccagtgtgcaaccatccagctggatttccagttgcccatcagatttaatcttacttatgtaagccatgatggtgatgataagaaaaggccagtgattgttcatcgagccatcttgggatcagtggaaagaatgattgctatcctcacagaaaactatgggggcaaatggcccttttggctgtcccctcgccaggtaatggtagttccagtgggaccaacctgtgatgaatatgcccaaaaggtacgacaacaattccacgatgccaaattcatggcagacattgatctggatccaggctgtacattgaataaaaagattcgaaatgcacagttagcacagtataacttcattttagttgttggtgaaaaagagaaaatcagtggcactgttaatatccgcacaagagacaataaggtccacggggaacgcaccatttctgaaactatcgagcggctacagcagctcaaagagttccgcagcaaacaggcagaagaagaattttaatgaaaaaattacccagattggctccatggaaaaggaggaacagcgtaccgtaaaattgactttgtactctgaaaacgtcaatttatattgaacttggaggagtttggcaaagtctgaataggtcaacctgcaggcgtaactattatgacctagtcagatttaaacaatgtgcatttgaaggagttaattaaaagagagccaataaaatgattttactcattcagtatctgagtactggaagtgaaacatgaggaatgctttagtgtaatgtgggagaacttttttgtaaatttaatgcaattgaaaaagttttcaaattcaattaagataactagaattggattatggtgtaaaaataaaaaaaaaatttattcacataaaaaaaaaaaaaaaaaaaaaaaa.SEQ ID NO: 2 A human TARS protein sequence is provided as GENBANK ™Accession No. P26639, whichis also the amino acid sequence encoded by SEQ IDNO: 1, which is set forth under GENBANK ™ Accession No. NM_152295MFEEKASSPSGKMGGEEKPIGAGEEKQKEGGKKKNKEGSGDGGRAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLKFEDEEAQAVYWHSSAHIMGEAMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLEALCKKIIKEKQAFERLEVKKETLLAMFKYNKFKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKALKIHKNSSTYWEGKADMETLQRIYGISFPDPKMLKEWEKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGAYIYNALIEFIRSEYRKRGFQEVVTPNIFNSRLWMTSGHWQHYSENMFSFEVEKELFALKPMNCPGHCLMFDHRPRSWRELPLRLADFGVLHRNELSGALTGLTRVRRFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEVWDQAEKQLENSLNEFGEKWELNSGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGDDKKRPVIVHRAILGSVERMIAILTENYGGKWPFWLSPRQVMVVPVGPTCDEYAQKVRQQFHDAKFMADIDLDPGCTLNKKIRNAQLAQYNFILVVGEKEKISGTVNIRTRDNKVHGERTISETIERLQQLKEFRSKQAEEEF. SEQ ID NO: 3 Mus musculus TARS polypeptidesequence having GENBANK ™ Accession No. Q9D0R2.MSQEKASSPSGKMDGEKPVDASEEKRKEGGKKKSKDGGGDGGRAELNPWPEYINTRLDMYNKLKAEHDSILAEKAAKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVVAKVNKVVWDLDRPLETDCTLELLKFEDEEAQAVYWHSSAHIMGEAMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLETLCKKIIKEKQTFERLEVKKETLLEMFKYNKFKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKTLKIHKNSSTYWEGKADMETLQRIYGISFPDPKLLKEWEKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGAYIYNTLMEFIRSEYRKRGFQEVVTPNIFNSRLWMTSGHWQHYSENMFSFEVEKEQFALKPMNCPGHCLMFDHRPRSWRELPLRLADFGVLHRNELSGALTGLTRVRRFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEIWNQAEKQLENSLNEFGEKWELNPGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGDDKKRPVIVHRAILGSVERMIAILTENYGGKWPFWLSPRQVMVVPVGPTCDEYAQKVRQQFHDAKFMADTDLDPGCTLNKKIRNAQLAQYNFILVVGEKEKASGTVNIRTRDNKVHGERTVEETVRRLQQLKQTRSKQAEEEF. SEQ ID NO: 4 C Elegans TARS polypeptide sequencehaving GENBANK ™ Accession No. P52709.MRLNCFRIFVHIQKPTQIFKPFYRSLSSEASDKYHFVNGHKMSKAPTDMAPWPAFIEERIKLWDKLKAEYDAEIAAKESEPIQITLPDGKIHEGKTWRTTPFEIAERISKGLAEAAVIAKVNGAVWDLDRPFEGNAKLELLKFDDDEAKQVFWHSSAHVLGEAMERYCGGHLCYGPPIQEGFYYDMWHENRTICPDDFPKIDQIVKAAVKDKQKFERLEMTKEDLLEMFKYNEFKVRIITEKIHTPKTTVYRCGPLIDLCRGPHVRHTGKVKAMAITKNSSSYWEGKADAESLQRLYGISFPDSKQLKEWQKLQEEAAKRDHRKLGKEHDLFFFHQLSPGSAFWYPKGAHIYNKLVDFIRKQYRRRGFTEVITPNMYNKKLWETSGHWQHYSEDMFKIEVEKEEFGLKPMNCPGHCLMFGHMPHTYNELPFRFADFGVLHRNEMSGALTGLTRVRRFQQDDAHIFCRQDQISEEIKQCLDFLEYAYEKVFGFTFKLNLSTRPEGFLGNIETWDKAEADLTNALNASGRKWVLNPGDGAFYGPKIDITIQDALKRNFQCATIQLDFQLPNQFDLSYFDEKGEKQRPVMIHRAVLGSVERMTAILTESYGGKWPFWLSPRQCKIITVHESVRDYANDVKKQIFEAGFEIEYEENCGDTMNKQVRKAQLAQFNFILVIGAKEKENGTVNVRTRDNAVRGEVALDKLISKFRRFADEYVADTEKSEEWA. SEQ ID NO: 5 S cerevisiae TARS polypeptidesequence having GENBANK ™ Accession No. P04801.MSASEAGVTEQVKKLSVKDSSNDAVKPNKKENKKSKQQSLYLDPEPTFIEERIEMFDRLQKEYNDKVASMPRVPLKIVLKDGAVKEATSWETTPMDIAKGISKSLADRLCISKVNGQLWDLDRPFEGEANEEIKLELLDFESDEGKKVFWHSSAHVLGESCECHLGAHICLGPPTDDGFFYEMAVRDSMKDISESPERTVSQADFPGLEGVAKNVIKQKQKFERLVMSKEDLLKMFHYSKYKTYLVQTKVPDGGATTVYRCGKLIDLCVGPHIPHTGRIKAFKLLKNSSCYFLGDATNDSLQRVYGISFPDKKLMDAHLKFLAEASMRDHRKIGKEQELFLFNEMSPGSCFWLPHGTRIYNTLVDLLRTEYRKRGYEEVITPNMYNSKLWETSGHWANYKENMFTFEVEKETFGLKPMNCPGHCLMFKSRERSYRELPWRVADFGVIHRNEFSGALSGLTRVRRFQQDDAHIFCTHDQIESEIENIFNFLQYIYGVFGFEFKMELSTRPEKYVGKIETWDAAESKLESALKKWGGNWEINAGDGAFYGPKIDIMISDALRRWHQCATIQLDFQLPNRFELEFKSKDQDSESYERPVMIHRAILGSVERMTAILTEHFAGKWPFWLSPRQVLVVPVGVKYQGYAEDVRNKLHDAGFYADVDLTGNTLQKKVRNGQMLKYNFIFIVGEQEMNEKSVNIRNRDVMEQQGKNATVSVEEVLKQLRNLKDEKRGDNVLA.SEQ ID NO: 6 Homo sapiens TARS cytoplasmic isoform 1 having GENBANK ™Accession No NP_689508.MFEEKASSPSGKMGGEEKPIGAGEEKQKEGGKKKNKEGSGDGGRAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLKFEDEEAQAVYWHSSAHIMGEAMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLEALCKKIIKEKQAFERLEVKKETLLAMFKYNKFKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKALKIHKNSSTYWEGKADMETLQRIYGISFPDPKMLKEWEKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGAYIYNALIEFIRSEYRKRGFQEVVTPNIFNSRLWMTSGHWQHYSENMFSFEVEKELFALKPMNCPGHCLMFDHRPRSWRELPLRLADFGVLHRNELSGALTGLTRVRRFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEVWDQAEKQLENSLNEFGEKWELNSGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGDDKKRPVIVHRAILGSVERMIAILTENYGGKWPFWLSPRQVMVVPVGPTCDEYAQKVRQQFHDAKFMADIDLDPGCTLNKKIRNAQLAQYNFILVVGEKEKISGTVNIRTRDNKVHGERTISETIERLQQLKEFRSKQAEEEF. SEQ ID NO: 7 is a portion of the sequence setforth in GENBANK ™ Accession No.: NM_152295.RAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELL K.SEQ ID NO: 8 is forward primer 5′caccagtgtgcaaccatccagctggatttccaggtgcccatcagatt taatc 3′.SEQ ID NO: 9 is reverse primer 5′gattaaatctgatgggccactggaaatccagctggatggttgcacac tggtg 3′.

DETAILED DESCRIPTION

Angiogenesis is involved in many cellular functions and processesincluding in diseases and conditions such as cancer, tumors,hemangiomas, vascular overgrowth, venous malformation, arterialmalformation, overweight (fat storage), macular degeneration,inflammatory disease, psoriasis, diabetes, and rheumatoid arthritis thatmay be characterized by excess angiogenesis and/or for which it may bedesirable to limit or reduce angiogenesis for treatment. Angiogenesis isalso involved in diseases and conditions such as tissue or organimplants, ischemia, cardiac infarction, tissue trauma, cartilage to bonetransformation, stroke, surgery, pregnancy, macular degeneration,vascular occlusion, which may be characterized by the presence ofinsufficient angiogenesis and/or for which it may be desirable toincrease angiogenesis as a treatment. It has now been identified thatthreonyl-tRNA synthetase (TARS) plays a role in angiogenesis and can beused in methods to diagnose and treat diseases and conditionscharacterized by abnormal (either increased or decreased) levels ofTARS. As used herein, with respect to TARS activity, the terms:“increased”, “elevated”, and “higher” are used interchangeably. As usedherein with respect to TARS molecule activity and quantitation, theterms “decrease”, “reduced”, and “lower” are used interchangeably.

In cancers, angiogenesis signaling can be an early step in invasivecancer growth, ascites formation, and metastasis. Cells that areenvironmentally stressed by hypoxia and/or starvation respond byexpressing genes that support anaerobic metabolism and stimulateangiogenesis. Because of their rapid growth, many cancer cell typescontinuously express these genes in an effort to continue growing in anutrient-poor environment. The development of vasculature, e.g.,angiogenesis, involves changes in protein synthesis and may be initiatedby environmental stress such as hypoxia or starvation in a cell.Aminoacyl tRNA synthetases are believed to function in some aspects ofangiogenesis. Cancer treatments that reduce angiogenesis have recentlybeen shown to causes hypoxia, enhancing the ability of cancer stem cellsto increase their invasiveness and metastatic potential. Hence, cancerdiagnoses and treatments that influence the hypoxic response aresignificant and novel area of cancer therapeutics.

It has now been identified that the determination of the levels andactivity of threonyl-tRNA synthetase (TARS) in cells and tissues can beused in methods to diagnose diseases and conditions in whichangiogenesis is altered, e.g., is abnormal compared to cells and tissueslacking the disease or condition. Thus, it is now understood that levelsof TARS expression and function can be useful in methods to diagnosisdiseases and conditions that are characterized at least in part byaltered TARS activity. Diseases and conditions that have altered TARSactivity may include angiogenesis-associated diseases and conditions,examples of which are provided herein, and include but are not limitedto cancer. Thus, the improved understanding the role of TARS in earlyangiogenesis signaling has now been used to identify novel diagnostictargets to recognize and diagnose angiogenesis-associated conditions,such as cancer, thus improving the likelihood of successful treatment.

Protein synthesis is known to include activities of aminoacyl tRNAsynthetases, which are enzymes that catalyze the aminoacylation of tRNAby their cognate amino acids. Threonyl-tRNA synthetase (TARS) is anaminoacyl tRNA synthetase that is known to charge tRNA with threonineduring protein synthesis. Protein synthesis plays a role in manydifferent activities of cells and tissues such as growth anddevelopment, differentiation, replication, signaling, etc. andalterations in aminoacyl tRNA synthetase activities and functions mayresult in disruption of cell processes and disease. One non-limitingexample of a disorder that TARS activity may play a role in is cancer.

Cancer cells respond to environmental stress and the tumormicroenvironment plays a role in determining cancer cell survival andgrowth responses. Cancer cells rely on these responses because theyrapidly outgrow their blood supply and must survive under conditions ofhypoxia, starvation, and metabolic stress. Cells relieve these stressesby decreasing protein translation through the unfolded protein responseand increasing blood supply through secretion of angiogenic cytokinesand growth factors. A novel connection between these metabolic andangiogenic responses has now been identified and features, in part, theability of tRNA synthetase inhibitors to alter the angiogenesissignaling pathway through a novel mechanism. In addition to having arole in cancer, angiogenesis also occurs physiologically during fetaldevelopment, wound healing, pregnancy, weight gain, and ischemicpreconditioning, and is a feature found numerous additional diseases andconditions.

TARS is an aminoacyl-tRNA synthetase that selectively catalyzes theATP-dependent formation of threonyl-tRNA, a substrate for the proteintranslation machinery. Aside from their canonical functions in proteinsynthesis, aminoacyl-tRNA synthetases have been implicated in autoimmuneand cytokine function, recovery from hypoxic stress, and angiogenesis.(Brown, M. V. et al. (2010) Vascul Pharmacol 52 (1-2), 21-26). Secretionand cytokine activities of extracellular TARS have now been examined andit has now been identified that TARS is secreted under conditions ofexposure to cytokines (e.g., TNF-αt and VEGF) and that one or morespecific domains of TARS, including the N-terminal domain of TARS (theTGS domain), are regulatory in nature and able to confer cytokineactivity.

Threonyl-tRNA synthetase (TARS) is a metabolic workhorse that functionsto charge tRNA with threonine during protein synthesis. TARS isubiquitously expressed in a number of prokaryotic and eukaryoticorganisms. TARS is alternatively known as threonine tRNA ligase 1;Threonine—tRNA ligase, threonyl-transfer ribonucleate synthetase,threonyl-transfer RNA synthetase, threonyl-transfer ribonucleic acidsynthetase, threonyl ribonucleic synthetase, threonine-transferribonucleate synthetase, threonine translase, TRS, and ThrRS. An exampleof a human TARS protein sequence is provided as GENBANK™ Accession No.P26639. Examples of TARS polypeptide sequences of other species include:Mus musculus: GENBANK™ Accession No.Q9D0R2; C Elegans: GENBANK™Accession No. P52709; S cerevisiae: GENBANK™ Accession No. P04801. Ahuman TARS nucleic acid sequence is provided as GENBANK™ Accession No.NM_152295.

It has now been discovered that TARS acts in a previously unknown mannerto promote angiogenesis. Studies have now shown that inhibition of TARSreduces both the hypoxic response of cancer cells and, unexpectedly,that application of exogenous purified TARS also stimulated angiogenesisin an in vivo angiogenesis assay. Thus, TARS may have dual functions asa metabolic regulator and as an angiogenic cytokine, and may be secretedfrom cells exposed to ischemic stress. It has also now been found thatTARS mRNA and TARS polypeptide may be selectively overexpressed invarious cancers, including but not limited to ovarian tumors, which arehighly angiogenic. An association between TARS polypeptide expressionand activity levels are positively correlated with angiogenesis and withcancer metastases. It has also now also been identified that levelsand/or activity of TARS mRNA and TARS polypeptide may be higher thannormal or lower than normal in additional diseases and conditions asdescribed herein.

It has now been shown that an increase in expression and/or activitiesof TARS (potentially including GTPase and Ap4A synthetic functions) iscorrelated with an increase in angiogenesis of cells and also that anincrease in expression and/or activity of TARS is correlated withmetastasis of a cancer compared to a lower level of expression and/oractivity of TARS in a poorly metastatic or non-metastatic cancer. Thus,expression and/or activity of TARS can be used to determine angiogenicpotential in cells and tissues and can also be used to determinemetastatic potential in cancers. In addition, the protein provides atarget for treatments to enhance or inhibit angiogenesis and fortreatments to inhibit the metastatic process in cancers.

Accordingly, the evaluation and comparison of levels of TARS molecules,such as TARS-encoding nucleic acids and TARS polypeptides, either normalor mutated, can be both diagnostic and prognostic for particulardiseases and conditions associated with angiogenesis. In some aspects ofthe invention, the disease or condition associated with angiogenesis iscancer and may be metastatic cancer. It has now been identified, that anelevated level of TARS, which may be a level of TARS polypeptide orlevel of TARS-encoding nucleic acid, for example, may indicate anincrease in angiogenesis in a cell, a tissue, and/or a subject. In someaspects, an increase in the TARS polypeptide or TARS-encoding nucleicacid level may indicate an increased likelihood for metastatic activityof a cancer, while lower levels of TARS and/or TARS activity mayindicate that the cancer has reduced metastatic potential. Further, bymonitoring a particular neoplastic growth over a period of time andcomparing changes in the level of a TARS polypeptide or TARS-encodingnucleic acid, one can evaluate changes in metastatic activity of thecancer.

The present invention provides methods of diagnosing a disease orcondition associated with abnormal TARS activity. As used herein, theterm “TARS activity” refers to a function of the TARS molecule, such as,but not limited to, aminoacylation of tRNA by threonine, association ofa TARS polypeptide with a von Hippel Lindau (VHL) polypeptide to form acomplex, association of a TARS polypeptide with elongation factor 1(eEF1) to form a complex, associate of a TARS polypeptide with an E3ubiquitin ligase, secretion of TARS protein or fragment of protein,binding of TARS to membrane receptors, or binding of TARS toextracellular matrix proteins.

In some embodiments of the invention, a disease or condition may becharacterized by increased TARS activity compared with a control levelof TARS activity. In certain embodiments of the invention a disorder orcondition may be characterized by decreased TARS activity compared witha control level of TARS activity. It will be understood that a change inTARS activity may be due to a change in the amount of TARS expressed ina cell, tissue, or subject, a change in the function or activity of TARSthat is expressed in a cell, tissue, or subject, and/or a change in thesecretion of TARS by a cell, tissue, or subject. Thus, in someembodiments of the invention, a reduction in TARS activity may be aresult of a reduction in the amount of TARS polypeptide in a cell,tissue, or fluid and in some embodiments the amount of TARS polypeptidemay be unchanged (e.g., normal compared with a normal control) but thefunctional activity of the TARS that is present in the cell, tissue, orsubject may be reduced. Similarly, in some embodiments of the invention,an increase in TARS activity may be a result of an increase in theamount of TARS polypeptide in a cell, tissue, fluid or subject and incertain embodiments the amount of TARS polypeptide may be unchanged(e.g., normal compared with a normal control) but the functionalactivity of the TARS that is present in a cell or tissue may beincreased. The altered activity may the result of an increase inavailability of a post-translationally modified version of TARS, whichmay be differentially secreted from one or more relevant cell types(including cancer cells, HUVEC cells, or cells of the innate immunesystem).

It has been identified that altered TARS activity in cells and/ortissues is correlated with various diseases and conditions. In certaindiseases and conditions the level of TARS activity is statisticallysignificantly higher in cells and/or tissues having the disease orcondition compared to the level of TARS activity in cells and/or tissuesthat do not have the disease or condition. A level of TARS activity in adisease or condition characterized by a significantly higher activitycompared to a normal control level may have a level of TARS activitythat is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 125%, 150%, 175%, 200%, or higher than a normal control level ofTARS activity, e.g., a level in an equivalent sample that does not havea disorder or condition characterized by elevated, e.g., higher levelsof TARS activity. As used herein, a disease or condition that may becharacterized by elevated TARS activity may also be referred to as adisease or condition associated with elevated TARS activity.

Examples of diseases and conditions that may be characterized elevatedTARS activity include, but are not limited to cancer, a tumor, ahemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, interstitial lung disease, and rheumatoidarthritis. An increase in angiogenesis may be a characteristic ofdiseases and conditions in which a higher TARS activity (versus a normalcontrol level) is present. Non-limiting examples of cancers that may becharacterized by elevated levels of TARS activity include a metastaticcarcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma;androgen independent prostate cancer; Kaposi's sarcoma; colorectalcancer, hepatobilliary cancer, gastric cancer, epithelial ovariancancer; lung cancer, and mesothelioma. In some aspects of the invention,assessing a change in the level of TARS activity in a disease orcondition characterized by increased TARS activity may be desirable, andmethods of the invention may be used to monitor the level of TARSactivity over time to assess changes.

In certain diseases and conditions a level of TARS activity isstatistically significantly lower in cells and/or tissues having thedisease or condition compared to the level of TARS activity in cellsand/or tissues that do not have the disease or condition. A level ofTARS activity in a disease or condition characterized by a significantlylower activity compared to a normal control level may have a level ofTARS activity that is at less than 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%, of alevel in a normal control of TARS activity, e.g., a level in anequivalent sample that does not have a disorder or conditioncharacterized by reduced, e.g., lower levels of TARS activity.

Examples of diseases and conditions that may be characterized by reducedTARS activity include, but are not limited to diseases or conditionssuch as is a tissue implant, organ implant, ischemia, cardiacinfarction, tissue trauma, cartilage to bone transformation, stroke,surgery, pregnancy, macular degeneration, or vascular occlusion. In someaspects of the invention, assessing a change in the level of TARSactivity in a disease or condition characterized by reduced (e.g., lowercompared to a control) TARS activity may be desirable and methods of theinvention may be used to monitor the level of TARS activity over time toassess changes. As used herein, a disease or condition that may becharacterized by reduced TARS activity may also be referred to as adisease or condition associated with reduced TARS activity.

Diagnostic methods of the invention may include determining a level ofTARS in a cell or tissue sample and comparing the determined level to acontrol level of TARS. For example a diagnostic test of the inventionmay include determining a level of TARS activity in a subject that has,is suspected of having, or is susceptible to having, or is at risk ofhaving a disease or condition characterized by altered TARS activity. ATARS level can be determined using methods of the invention to measurethe amount and/or activity of a TARS molecule in an in vitro assay of abiological sample that has been obtained from the subject. As usedherein, the term “measure” may refer to a determination of the presenceor absence of a TARS molecule, may refer to a determination of aquantity a TARS molecule, or may refer to a determination of an activitylevel of a TARS molecule. Methods of measuring polypeptides or nucleicacids are known in the art, and non-limiting examples of measuring meansare provided herein.

Detection methods suitable for use in methods of the present inventioncan be used to detect TARS polypeptide or nucleic acid molecules in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of TARS mRNA include reverse transcriptasequantitative polymerase chain reaction (RT-qPCR), Northernhybridizations, in situ hybridizations, DNA or oligonucleotide array,and next generation sequencing. In vitro techniques for detection ofTARS DNA include polymerase chain reaction (PCR) and Southernhybridizations. In vitro techniques for detection of TARS polypeptideinclude, but are not limited to enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence,and other known suitable techniques. Alternatively, TARS polypeptide canbe detected in vivo in a subject by introducing into the subject alabeled anti-TARS antibody. For example, the antibody can be labeledwith a detectable marker such as a colorimetric marker, enzymaticmarker, radioactive marker, etc. whose presence and location in asubject can be detected by standard imaging techniques.

Certain embodiments of the invention include methods of performing an invivo diagnostic test in a subject. An in vivo diagnostic test of theinvention may include administering to a subject one or more compoundsuseful to determine a level of TARS in a cell or tissue in the subject.In a non-limiting example, a detectably labeled antibody or antibodyfragment that binds a TARS polypeptide can be administered to thesubject and can be measured to determine the TARS level in the subject,e.g., in a cell, or tissue in the subject. It will be understood thatwhen using an in vivo method of the invention, the biological sampletested from the subject may comprise an in vivo cell or tissue sampleand that the in vivo cell or tissue is considered to have been obtainedfrom the subject even though the sample is not removed from the subjectprior to use in the diagnostic method of the invention.

In some aspects of the invention, a disease for diagnosis may be one forwhich a biological sample can be obtained from a subject for testing andassay. In certain embodiments, a diagnosis is done in vivo and thediagnostic molecule (e.g., antibody, binding molecule or other compound)that is used to detect a TARS molecule must be administered to asubject. Where the present invention provides for the administration of,for example, antibodies or one or more other detectable compounds orsmall molecules to a subject, then this may be by any suitable route.The route of administration will depend, in part, on the location of thedisease or condition for diagnosis. For example, if the disease to bediagnosed in vivo includes a tumor, an appropriate method ofadministration may be by injection directly to the site of the tumor.Thus, administration may by injection, including subcutaneous,intramuscular, intravenous, intrathecal, intracranial, and intradermalinjections. Administration can also be by other means as describedelsewhere herein. Administration via a catheter is also a mode ofadministration useful in some embodiments of the invention.

In Vivo Imaging Techniques

A molecule used in a diagnostic method of the invention, (e.g., ananti-TARS antibody or fragment thereof, a small molecules that bind toTARS or to TARS in associated with another polypeptide, etc.—(alsoreferred to herein as “a diagnostic molecule of the invention”) may alsobe used for imaging purposes, for example, to detect tumor metastasis.Suitable labels that may be attached to a diagnostic molecule and usedin methods of the invention include, but are not limited to,radioisotopes, iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium(³H), indium (¹¹²In), and technetium (⁹⁹mTc), fluorescent labels, suchas fluorescein and rhodamine, and biotin, and nano- or micro-particles.

In some embodiments a diagnostic molecule (e.g., an antibody or fragmentthereof etc.) used for a diagnostic method of the invention may belabelled, or otherwise modified, to permit detection. Such labeleddiagnostic molecules can be used for real-time in vivo diagnostics usingsample that remains within (e.g., is not removed from) a subject or forin vitro diagnostics using a sample that is removed from a subject.Although not intending to be limited, an example of an in vitrodiagnostic method of the invention may include use of a labeledanti-TARS antibody to detect tumor margins in tissue, for example, in atissue removed during the process of surgery. Detectable labels that canbe used in conjunction with a diagnostic molecule of the invention maybe any that do not substantially interfere with the diagnostic moleculebinding to a TARS molecule, but that allow external detection. Examplesof detectable labels and methods suitable for use in in vitro diagnosticmethods of the invention are described in detail elsewhere herein.Suitable in vivo detectable labels may include those that may bedetected by X-radiography, NMR or MRI. For X-radiographic techniques,suitable detectable labels include any radioisotope that emitsdetectable radiation but that is not overtly harmful to the patient,such as barium or cesium, for example. Suitable detectable labels forNMR and MRI generally include those with a detectable characteristicspin, such as deuterium, which may be incorporated into the antibody bysuitable labeling of nutrients for the relevant hybridoma, for example.

The size of the subject, and the imaging system used, will determine thequantity of imaging moiety needed to produce in vivo diagnostic images.In the case of a radioisotope moiety, for a human subject, the quantityof radioactivity injected will normally range from about 5 to 20millicuries of technetium-99m. The labeled diagnostic molecule (forexample, labeled antibody or antibody fragment thereof or other TARSbinding molecule) will then preferentially accumulate at the location ofsample cells that contain TARS. The labeled diagnostic molecule can thenbe detected using known techniques.

In some embodiments of the invention, a diagnostic molecule for use inan in vitro or in vivo diagnostic method of the invention may beadministered to a subject. For example, a diagnostic molecule may beadministered to a subject and subsequently a biological sample may beobtained from that subject and examined in vitro using a diagnosticmethod set forth herein. In certain embodiments, a diagnostic moleculemay be administered to a subject and subsequently an in vivo testingmethod used to determine a TARS activity level in a biological samplethat remains within the subject. Thus, in some aspects of the invention,a diagnostic molecule may be administered orally, topically, or byparenteral means, including subcutaneous and intramuscular injection,implantation of sustained release depots, intravenous injection,intrathecal injection, intranasal administration, vaginaladministration, rectal administration, and the like. Accordingly, adiagnostic molecule of the invention may be administered as apharmaceutical composition comprising the treatment compound incombination with a pharmaceutically acceptable carrier. Suchcompositions may be aqueous solutions, emulsions, creams, ointments,suspensions, gels, liposomal suspensions, and the like. Suitablecarriers (excipients) include, but are not limited to, water, saline,Ringer's solution, dextrose solution, and solutions of ethanol, glucose,sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol(PEG), phosphate, acetate, gelatin, collagen, vegetable oils, and thelike. One or more pharmaceutically acceptable salts may be included in apharmaceutical composition of the invention. Exemplary pharmaceuticallyacceptable salts include, but are not limited to mineral acid salts suchas hydrochlorides, hydrobromides, phosphates, sulfates, and the like;and the salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. One may additionally include suitablepreservatives, stabilizers, antioxidants, antimicrobials, and bufferingagents, for example, BHA, BHT, citric acid, ascorbic acid, tetracycline,and the like. Cream or ointment bases useful in formulation includelanolin and the like. Other topical formulations include aerosols,bandages, and other wound dressings or wound-packing materials.

The amount of a diagnostic molecule required for a diagnostic method ofthe invention will of course vary depending upon the nature, location,and severity of the disorder, the age and condition of the subject, andother factors readily determined by one of ordinary skill in the art,including, but not limited to a health-care professional.

A method of the invention may include comparing a level of a TARSmolecule in a sample to a control value or level for the TARS molecule.As used herein a “control” may be a normal control or a disease control.Selection and use of appropriate controls in diagnostic methods are wellknown in the art. In some embodiments of the invention, a normal controllevel may be obtained from a cell or tissue sample that is known to befree of the disease or condition associated with altered (increased ordecreased) TARS activity. A normal control level of a TARS moleculeand/or TARS molecule activity can readily be determined by measuring alevel of the TARS molecule or TARS molecule activity using an assayprovided herein and/or any suitable assay available in the art. In someembodiments of the invention, a disease control level may be obtainedfrom a cell or tissue sample that includes cells and/or tissues knownhave the disease or condition associated with altered TARS activity. Insome embodiments, a disease control TARS level may be based on levelsobtained from one or more subjects known to have the disease orcondition associated with altered TARS activity. In certain embodimentsof the invention, the disease control may be a sample from a subjectdiagnosed with the disease or condition and the subject's diseasecontrol may be compared to another sample obtained from the subject at adifferent time. Disease control levels of TARS and TARS activity canreadily be determined by measuring levels of TARS or TARS activity,respectively, in a biological sample of individuals having the diseaseor condition associated with altered (higher or lower) TARS activity.

In some aspects of the invention methods are provided that includecomparing a level of TARS determined or measured a sample obtained froma subject to a control value for determining a disease stage or subjectprognosis. In addition, onset, progression, or regression of a diseaseor condition characterized by altered TARS activity can be assessed bydetermining TARS levels in a subject by measuring TARS levels in samplesobtained from or tested in the subject at two, three, four, five, ormore different times. In a method that utilizes two or more samplesobtained from a subject at different times, values obtained from asample obtained at one time can be compared to values obtained at othertimes. For example, a first level obtained from the subject may serve asa baseline level or control level for that subject, thus allowingcomparison of the TARS level and the determination of change orstability of the TARS level over time. TARS levels could also bemeasured after a specific course of treatment against cancer or otherdiseases has been initiated, with the intent of determining the efficacyof that treatment or the onset of relapse as a consequence of resistanceto the treatment.

The status of the angiogenesis-associated disease or condition can bemonitored using methods of determining TARS polypeptide activity orlevels of nucleic acids that encode a TARS polypeptide, etc. In someaspects of the invention, a desired response to treatment of theangiogenesis-associated disease or condition also can be delaying theonset or even preventing the onset of the angiogenesis-associateddisease or condition.

The invention, in some aspects, includes methods and assays (e.g.binding assays, gel electrophoresis; mass spectrometry; NMR; etc.) todetermine changes in TARS level and/or activity in a subject or cellsample (e.g., cell culture) over time. This allows monitoring of TARSlevels and/or activity in a subject who is to undergo treatment for acancer or other proliferative disease or condition and also enables tomonitoring in a subject who is currently undergoing therapy for thecancer or proliferative disease or condition. Thus, methods of theinvention may be used to diagnose or assess a cancer or proliferativedisease or condition in a subject and may also be used to assess theefficacy of a therapeutic treatment of a cancer or proliferative diseaseor condition and for assessment of the activity or level of a TARSmolecule in a subject at various time points. For example, a subject'sTARS level and/or activity can be determined prior to the start of atherapeutic regimen (either prophylactic or as a treatment of a canceror other proliferative disease or condition), during the treatmentregimen and/or after a treatment regimen, thus providing information onthe status of the cancer or proliferative disease or condition in thesubject.

Assessment of efficacy of candidate TARS-modulating compounds toincrease or decrease expression of TARS polypeptide-encoding nucleicacid or a TARS polypeptide in a cell or tissue may also be done usingassays of the invention in cells from culture—e.g., as screening assaysto assess candidate TARS-modulating compounds to modulate TARSpolypeptide activity. TARS-modulating compounds that alter TARSpolypeptide activity in a cell, tissue, or subject may be used in thetreatment of a cancer or proliferative disease or condition or as apretreatment for a cancer or proliferative disease or condition (e.g.,to prepare a cell or subject for subsequent treatment).

It will be understood that a therapeutic regimen may be eitherprophylactic or a treatment of a cancer or proliferative disease orcondition in a subject. The invention in some aspects provides methodsthat may be used to monitor a subject's response to prophylactic therapyand/or treatment for a cancer or proliferative disease or conditionprovided to a subject. Methods of the invention (e.g. binding assays,gel electrophoresis; mass spectrometry; NMR; etc.) may also be useful tomonitor the onset, progression, or regression of cancer or proliferativedisease or condition in a subject at risk of developing the cancer orproliferative disease or condition. TARS polypeptide levels and/oractivity or TARS-encoding nucleic acid levels may be determined in two,three, four, or more biological samples obtained from a subject atseparate times. The TARS polypeptide levels and/or activity or theTARS-encoding nucleic acid levels determined in the samples may becompared and changes in the levels and/or activity over time may be usedto assess the status and stage of a cancer or proliferative disease orcondition in the subject (or in a cell or tissue sample) and/or theeffect of a treatment strategy on the cancer or proliferative disease orcondition in a subject (or a cell or tissue sample). Some embodiments ofmethods of the invention can be used to assess treatments for cancer orproliferative disease or conditions and can be used to obtain usefulprognostic information by providing an indicator of a status of a canceror proliferative disease or condition and in some embodiments of theinvention, can be used to select a therapy for the subject, for example,to select a drug therapy, behavioral therapy, surgical therapy, etc.

Assays for assessing TARS levels in embodiments of the invention mayinclude determining one or more TARS levels and/or activities, includingbut not limited to determining levels of nucleic acids that encode TARSpolypeptides and/determining levels of TARS polypeptides in cells,tissues, and subjects. Levels of TARS polypeptide-encoding nucleic acidsand TARS polypeptides can be determined in a number of ways whencarrying out the various methods of the invention. In some embodimentsof the invention, a level of a TARS polypeptide-encoding nucleic acid orTARS polypeptide is measured in relation to a control level of TARSpolypeptide-encoding nucleic acid or TARS polypeptide, respectively, ina cell, tissue, or subject. One possible measurement of the level ofTARS polypeptide-encoding nucleic acid or polypeptide is a measurementof an absolute level of TARS polypeptide-encoding nucleic acid or TARSpolypeptide. This could be expressed, for example, in the level of TARSpolypeptide-encoding nucleic acid or polypeptide per unit of cells ortissue. Another measurement of a level of TARS polypeptide-encodingnucleic acid or TARS polypeptide is a measurement of the change in thelevel of the TARS polypeptide-encoding nucleic acid or TARS polypeptideover time. This may be expressed in an absolute amount or may beexpressed in terms of a percentage increase or decrease over time.Antibodies or antigen-binding fragments or other compounds thatspecifically bind a TARS polypeptide or a nucleic acid that encodes aTARS polypeptide may be used in embodiments of methods of the inventionto assess TARS polypeptide and TARS polypeptide-encoding nucleic acidmolecules to assess the status of a cancer or proliferative disease orcondition and/or the efficacy of treatments for cancer or otherproliferative disease or condition.

The present invention, in some aspects, provides methods of determininga prognosis in a subject that has a disease or condition associated withan altered TARS activity. A prognosis for a subject having such adisease or condition can be determined by measuring levels of TARS in abiological sample obtained from a subject to be tested. Expressionand/or activity of TARS in the biological sample that is greater than acontrol level indicates a worse prognosis for the subject. Such methodscan be used to assess the prognosis of a disease or condition associatedwith altered TARS activity. Examples of prognoses may include, but arenot limited to, determination of the likelihood of metastases in acancer, the likelihood of recovery from or remission in the disease orcondition, the likelihood of progression of the disease or condition.

The present invention further provides methods for determining themetastatic potential of a cancer and/or tumor by measuring the level ofTARS expression and/or activity in a biological sample of the cancer,tumor or fluid sample that is obtained from the subject. Expressionand/or activity of TARS in the cancer tissue or fluid that is greaterthan a control level for that particular cancer or tumor tissue mayindicate an increased metastatic potential of that cancer or tumor inthe subject. In some embodiments, a greater level of TARS in a samplefrom a subject indicates versus a control level correlates with anincreased risk of metastases of the cancer in the subject. Thus, anincrease in a level of TARS in a subject known to have cancer that is atleast 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%,150%, or 200%, above a level that is diagnostic for the cancer mayindicate an increased likelihood and risk of metastasis of the cancer inthe subject and angiogenic status of the tumor. This type of informationcan be used by a health-care professional to help determine a medicallyappropriate course of treatment for the subject. For example, adetermination that a subject is at higher risk of metastases from acancer may suggest use of a more aggressive treatment of the cancer thanif a lower risk of metastases is determined. Thus, for example, if anelevated level of TARS is measured in a subject known to have prostatecancer and the level is beyond that which is diagnostic for the canceritself, this may indicate increased risk that the cancer willmetastasize in the subject. Such an indication determined in a subjectwith a cancer, (e.g., ovarian cancer, prostate cancer, etc.) mayindicate a need for more aggressive surgical treatment, radiationtreatment, and/or chemotherapy treatment than if an increased level ofTARS activity is not present. In addition, a measure of the angiogenicstate of the tumor could provide information related to the use ofanti-angiogenic treatment in patients that exhibit higher than normalTARS in their tumor or fluid samples.

Changes in a subject's condition can also be monitored using methods ofthe present invention by comparing changes in TARS levels and/oractivity in cells and tissues in biological samples obtained from thesubject at two or more time points. Thus, a first level of TARS can bedetermined in a sample obtained from a subject at a first time point anda second level of TARS can be determined in a subsequent sample obtainedfrom the subject at a second time point and the levels in the samplescan be compared. For a disorder characterized by higher TARS activity, alower TARS level in a first sample compared to the level in a secondsample from the same subject can be used to determine the onset orprogression of a disease or condition and a higher TARS level in thefirst sample compared to the level in the second sample can indicateregression of the disease or condition. Similarly, for a disordercharacterized by lower TARS activity, a higher TARS level in a firstsample compared to the level in a second sample from the same subjectcan be used to determine the onset or progression of a disease orcondition and a lower TARS level in the first sample compared to thelevel in the second sample can indicate regression of the disease orcondition in the subject. Such information about the stage or status ofthe disease or condition can be used to assist a health-care provider toselect a treatment for administration to the subject or can be used by ahealth-care professional to adjust (e.g., increase, decrease, or stop) atreatment that is being provided to the subject.

As used herein a “subject” refers to any warm-blooded animal, such as,but not limited to a human, a non-human primate, a rodent, a dog, cat,or other animal. Thus, in addition to human medical application, someaspects of the invention include veterinary application of methodsdescribed herein. A subject may be known to have a disease or conditioncharacterized by an altered TARS activity as compared to a controllevel, and thus may be a subject diagnosed with the disease orcondition. In some embodiments, a subject may not have been previouslyor currently diagnosed with such a disease or condition, but may beconsidered to be at risk of for having the disease or condition, forexample, a subject who may be free of a detectable disease or conditionin which TARS activity is altered. In some embodiments of the invention,a subject may have previously been diagnosed with a disease, for examplediagnosed with a cancer, but the subject may be in remission at the timea diagnostic test is performed using methods of the invention.

In some embodiments of the invention, a biological sample comprises acell or tissue or extracellular material from a subject. In someembodiments a sample is a tumor sample. A tissue sample or tumor samplemay comprise tissue or a suspension of cells. A tissue section, forexample, a freeze-dried, paraffin embedded, or fresh frozen section oftissue removed from a subject, or a section of a tumor biopsy can beused as the biological sample. Moreover, a biological sample may be abiological fluid obtained from a subject (e.g., blood, Aqueous humourand vitreous humour, bile, blood, serum, breast milk, cerebrospinalfluid, lymph, female or male ejaculate, gastric fluid, mucus, peritonealfluid, plural fluid, saliva, sebum, semen, sweat, tears, vaginalsecretion, urine, ascites, spinal fluid, etc.). Following collection,fluids, cells, tissues, tumor or other biological samples can be storedat temperatures below −20° C. to prevent degradation until the detectionmethod is to be performed. In some embodiments of the invention, abiological sample in which a TARS molecule is to be detected is aprostate or ovarian tissue and/or tumor sample. In certain embodimentsof the invention, a biological sample in which TARS mRNA or TARSpolypeptide is to be detected is, for example, a prostate, colon,cervical, ovarian, or other tumor. In some aspects of the invention, abiological sample may comprise TARS that has been secreted from the cellin which it was produced. In certain aspects of the invention, abiological sample may comprise a TARS molecule that is a non-secretedmolecule, which as used herein, is a TARS molecule that was produced ina cell but not secreted by that cell into the extracellular environment.

Biological samples for use in methods of the invention (e.g., fordiagnostic purposes) may be obtained from any number of sources. Asample obtained directly from a cancer or tumor, such as the stroma orcytosol, may be used to determine the metastatic potential of the canceror tumor. Such diagnostic methods may be useful to monitor progress of asubject, such as after surgery to remove a tumor. If a reference (e.g.,control or baseline) TARS level determination is made in a sample afterthe operation, and one or more additional determinations are made insamples taken from the subject at later times, any increase in the levelof TARS could be indicative of a relapse, or possibly a metastasis.

As used herein, the term “isolated”, when used in the context of abiological sample, is intended to indicate that the biological samplehas been removed from a subject. In some embodiments of the invention, abiological sample comprises a sample that has been isolated from asubject and is subjected to a method of the present invention withoutfurther processing or manipulation subsequent to its isolation. In someembodiments of the invention, a biological sample can be processed ormanipulated subsequent to being isolated and prior to being subjected toa method of the invention. For example, a sample can be refrigerated(e.g., stored at 4° C.), frozen (e.g., stored at −20° C., stored at−135° C., frozen in liquid nitrogen, or cryopreserved using any one ofmany standard cryopreservation techniques known in the art).Furthermore, a sample can be purified subsequent to isolation from asubject and prior to subjecting it to a method of the present invention.

As used herein, the term “purified” when used in the context of abiological sample, is intended to indicate that at least one componentof the isolated biological sample has been removed from the biologicalsample such that fewer components, and consequently, purer components,remain following purification. For example, a serum sample can beseparated into one or more components using centrifugation techniquesknown in the art to obtain partially-purified sample preparation.Furthermore, it is possible to purify a biological sample such thatsubstantially only one component remains. For example, a tissue or tumorsample can be purified such that substantially only the polypeptide ormRNA component of the biological sample remains.

Furthermore, it may be desirable to amplify a component of a biologicalsample such that detection of the component is facilitated. For example,the mRNA component of a biological sample can be amplified (e.g., byRT-PCR) such that detection of TARS mRNA is facilitated. As used herein,the term “RT-PCR” (an abbreviation for reverse transcriptase-polymerasechain reaction) involves subjecting mRNA to the reverse transcriptaseenzyme, resulting in the production of a cDNA which is complementary tothe base sequences of the mRNA. Large amounts of selected cDNA can thenbe produced by means of the polymerase chain reaction which relies onthe action of heat-stable DNA polymerase for its amplification action.Alternative amplification methods include: self-sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87: 1874-1878), transcriptional amplification system (Kwoh, D. Y. etal., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase(Lizardi, P. M. et all, 1988, Bio/Technology 6: 1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

TARS Detection Techniques

TARS molecules (for example, TARS polypeptides and nucleic acids thatencode TARS polypeptides), can be detected and measured using anysuitable means known in the art. In some embodiments of the invention, adetection or measurement means for TARS molecules includes animmunological assay, nucleotide determination (mRNA or DNA), massspectrometry assessment, TARS aminoacylation assay, TARS active sitedetermination assay, or a TARS binding assay that may include aTARS-binding reporter molecule. Examples of immunological assayssuitable for use in methods of the invention may include, but are notlimited to ELISA assays, assays that utilize an anti-TARS antibody (orFV derivative) to which is conjugated a detectable label, (examples ofwhich include but are not limited to a radiolabel, non-limiting examplesof which are technicium and indium). In some aspects of the invention,TARS levels may be measured in complex mixtures using an amino acid(threonine) activation assay, aminoacylation assay, or binding of athreonine specific tRNA, or a nucleic acid aptamer designed and selectedto bind to threonyl-tRNA synthetase.

In some embodiments of the invention, levels of a TARS polypeptide maybe detected in complex protein mixtures using mass spectrometry methods,which may include a TARS-specific peptide as an internal standard toallow quantitation. Methods of measuring levels of nucleic acidsencoding TARS (i.e. TARS mRNA) may include, but are not limited to,real-time polymerase chain reaction (qRT-PCR), DNA array, and nextgeneration sequencing methods.

The present invention features agents that are capable of detectingand/or quantitating a TARS polypeptide or a TARS-encoding nucleic acidsuch that the presence and/or level of TARS are determined. As definedherein, an “agent” refers to a substance that is capable of identifyingor detecting TARS in a biological sample (e.g., identifies or detectsTARS mRNA, TARS DNA, TARS polypeptide, TARS activity, etc.). In someembodiments of the invention, the agent is a labeled or a labelableantibody or molecule (e.g., a binding partner) that specifically bindsto a TARS polypeptide. It will be understood that as used herein, theterm “polypeptide” is used in reference to an amino acid sequence of afull-length TARS protein or a portion of a TARS protein. As used herein,the terms “labeled” or “labelable” refers to the attaching or includingof a label (e.g., a marker or indicator) or ability to attach or includea label (e.g., a marker or indicator). Markers or indicators useful inmethods of the invention may include, but are not limited to, forexample, radioactive molecules, colorimetric molecules, and enzymaticmolecules that produce detectable changes in a substrate.

In some embodiments of the invention, an agent is an antibody thatspecifically binds to all or a portion of a TARS polypeptide. As usedherein, the phrase “specifically binds” refers to binding of, forexample, an antibody to an epitope or antigen or antigenic determinantin such a manner that binding can be displaced or competed with a secondpreparation of identical or similar epitope, antigen or antigenicdeterminant. In an exemplary embodiment, the agent is an antibody thatspecifically binds to all or a portion of the human TARS polypeptide. Insome embodiments of the invention, an ELISA is used in conjunction withthe antibody to determine the presence and/or level of TARS polypeptidein a biological sample. Methods of the invention for detecting thepresence and/or quantity of a TARS molecule may also include proceduressuch as an immunological assay, a polymerase chain reaction, real-timepolymerase chain reaction (qRT-PCR), mass spectrometry, a TARSaminoacylation assay, TARS active site determination assay, or a TARSbinding assay comprising a TARS-binding reporter molecule. In addition,embodiments of the invention include may include nucleic “aptamers”,i.e. nucleic acids (DNA, RNA or peptide nucleic acids [PNAs]) thatpossess high affinity for TARS derived polypeptides and can be readilylabeled for high throughput binding assays. Aptamers can be produced bystandard molecular biological techniques by those skilled in the art byrepeated rounds of binding, selection, and affinity, and amplification(Hamaguchi, et al. Anal. Biochem. (2001) 294; pt 2, pages 126-131).

In some embodiments of the invention an agent is a labeled or labelablenucleic acid probe capable of hybridizing to a TARS nucleic acid, (e.g.,a TARS RNA or DNA). For example, the agent can be an oligonucleotideprimer for the polymerase chain reaction that flanks or lies within thenucleotide sequence encoding human TARS. In some embodiments of theinvention, the biological sample being tested is an isolate, forexample, RNA. In yet another embodiment, the isolate (e.g., the RNA) issubjected to an amplification process that results in amplification ofTARS nucleic acid. As defined herein, an “amplification process” isdesigned to strengthen, increase, or augment a molecule within theisolate. For example, where the isolate is mRNA, an amplificationprocess such as RT-PCR can be utilized to amplify the mRNA, such that asignal is detectable or detection is enhanced. Such an amplificationprocess is beneficial particularly when the biological, tissue, or tumorsample is of a small size or volume.

TARS Nucleic Acid Binding Agents

Types of agents that can be used to determine levels of TARS-encodingnucleic acids may include, but are not limited to cDNA, riboprobes,synthetic oligonucleotides and genomic probes. The type of agent (e.g.probe) used will generally be dictated by the particular situation, suchas riboprobes for in situ hybridization, and cDNA for Northern blotting,for example. Most preferably, the probe is directed to nucleotideregions unique to the polypeptide. Detection of the TARS-encoding gene,per se, will be useful for diagnostic methods of the invention and forscreening for mutations associated with enhanced expression. Other formsof assays to detect targets more readily associated with levels ofexpression—transcripts and other expression products—will generally beuseful as well. A probe may be as short as is required to differentiallyrecognize TARS mRNA transcripts, and may be as short as, for example, 15bases; however, probes of at least 17 bases, 18 bases, 19, bases, 20bases, or more may be used.

A probe may also be reverse-engineered by one skilled in the art, forexample using the amino acid sequence of GENBANK™ AccessionNo.:NM_152295. However use of such probes may be more limited than thenative DNA sequence, as it will be appreciated that any one givenreverse-engineered sequence will not necessarily hybridize well, or atall, with any given complementary sequence reverse-engineered from thesame peptide, owing to the degeneracy of the genetic code. This is afactor common in the calculations of those skilled in the art, and thedegeneracy of any given sequence is frequently so broad as to yield alarge number of probes for any one sequence.

The form of labeling of a probe used in an embodiment of the inventionmay be any that is appropriate, such as the use of radioisotopes, forexample, ³²P and ³⁵S, etc. Labeling with radioisotopes may be achieved,whether the probe is synthesized chemically or biologically, by the useof suitably labeled bases using methods well known in the art.

TARS RNA Detection Techniques

Detection of RNA transcripts may be achieved by Northern blotting, forexample, wherein a preparation of RNA is run on a denaturing agarosegel, and transferred to a suitable support, such as activated cellulose,nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA isthen hybridized to the preparation, washed and analyzed byautoradiography.

Detection of RNA transcripts can further be accomplished using knownamplification methods. For example, it is within the scope of thepresent invention to reverse transcribe mRNA into cDNA followed byreal-time polymerase chain reaction (RT-PCR); or, to use a single enzymefor both steps as described in U.S. Pat. No. 5,322,770, or reversetranscribe mRNA into cDNA followed by symmetric gap ligase chainreaction (RT-AGLCR). Each of these methods is well known and routinelyused in the art. Other known amplification methods can also be utilizedin methods of the invention, including, but not limited to:

In situ hybridization visualization may also be employed, wherein aradioactively labeled antisense RNA probe is hybridized with a thinsection of a biopsy sample, washed, cleaved with RNase and exposed to asensitive emulsion for autoradiography. Biological samples may bestained with haematoxylin to demonstrate the histological composition ofthe sample, and dark field imaging with a suitable light filter showsthe developed emulsion. Non-radioactive labels such as digoxigenin, etc.may also be used.

TARS Antibodies and Additional Binding Agents

It will be appreciated that antibodies for use in accordance withdiagnostic methods of the present invention may be monoclonal orpolyclonal as appropriate. Antibody equivalents of these may comprise:the Fab′ fragments of the antibodies, such as Fab, Fab′, F(ab′)2 and Fv;idiotopes; or the results of allotope grafting (where the recognitionregion of an animal antibody is grafted into the appropriate region of ahuman antibody to avoid an immune response in the patient), for example.Single chain antibodies may also be used. Other suitable modificationsand/or agents will be apparent to those skilled in the art. Chimeric andhumanized antibodies are also within the scope of the invention and avariety of approaches for making chimeric antibodies are known in theart. Additionally, a chimeric antibody can be further “humanized” suchthat parts of the variable regions, especially the conserved frameworkregions of the antigen-binding domain, are of human origin and only thehypervariable regions are of non-human origin. Such alteredimmunoglobulin molecules may be made by any of several techniques knownin the art.

In addition to using antibodies to bind to and for detection of a TARSmolecule in diagnostic methods of the invention, it may also be possibleto use other molecules or compounds that bind to a TARS molecule indiagnostic methods. For example, it may be possible to identifyantagonists, compounds, and/or molecules such as polypeptides thatspecifically bind to a TARS molecule. In addition, it may also bepossible to use an antibody or other compound or molecule that binds to,and permits detection of a TARS molecule in a biological sample. In someembodiments a TARS molecule will detected as part of a complex with oneor more additional polypeptides. One non-limiting example of a bindingmolecule that may be useful in methods of the invention is a von HippelLindau (VHL) polypeptide. VHL polypeptides bind to and form a complexwith TARS, and in some embodiments of the invention may be used as anagent to detect the presence and/or to quantify a TARS molecule in abiological sample. Other polypeptides include, but are not limited to,eukaryotic elongation factor EF1A1, or Poly ADP ribose polymerase(PARP), as shown in FIG. 16.

An isolated TARS polypeptide, or fragment thereof, can be used as animmunogen to generate antibodies that bind TARS using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length TARS polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of TARS for use asimmunogens. The antigenic peptide of TARS may comprise at least 8 aminoacid residues of the amino acid sequence shown in GENBANK™ AccessionNo.:NM_152295 and encompasses an epitope of TARS such that an antibodyraised against the peptide forms a specific immune complex with TARS.Polypeptides that may be used as immunogens include but are not limitedto the sequence set forth as SEQ ID NO:7RAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESWKTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLK, which is a portion of thesequence set forth in GENBANK™ Accession No.:NM_152295. In some aspectsof the invention, an antigenic peptide may comprise at least 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or more residues. Antigenic polypeptides comprising at least 50, 100,150, 200 or 250 amino acid residues are also within the scope of thepresent invention. Preferred epitopes encompassed by the antigenicpeptide are regions of TARS that are located on the surface of thepolypeptide, e.g., hydrophilic regions.

A TARS immunogen typically may be used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for examples, recombinantly expressed TARS polypeptide or achemically synthesized TARS polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic TARS preparation induces a polyclonal anti-TARSantibody response. The immunogen may further include a portion ofnon-TARS polypeptide, for example, a polypeptide useful to facilitatepurification.

Accordingly, another aspect of the invention pertains to the use ofanti-TARS antibodies to determine the level and activity of TARSpolypeptides. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as TARS. The invention may include use of polyclonal and monoclonalantibodies that bind TARS. The term “monoclonal antibody” or “monoclonalantibody composition”, as used herein, refers to a population ofantibody molecules that contain only one species of an antigen bindingsite capable of immunoreacting with a particular epitope of TARS. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular TARS polypeptide with which it immunoreacts.

Polyclonal antibodies generated by the above or another technique may beused directly, or suitable antibody producing cells may be isolated fromthe animal and used to form a hybridoma by known means [Kohler andMilstein, Nature 256:795. (1975)]. Selection of an appropriate hybridomawill also be apparent to those skilled in the art, and the resultingantibody may be used in a suitable assay to identify and/or quantify aTARS molecule.

TARS Protein Detection Techniques

Methods of the invention may include the use of diagnostic molecules(e.g., antibodies, antibody equivalents, binding molecules, etc.) todetect TARS polypeptides. Methods for the detection of polypeptides arewell known to those skilled in the art, and include ELISA (enzyme linkedimmunosorbent assay), RIA (radioimmunoassay), Western blotting, andimmunohistochemistry. Methods for immunoassays are routinely used andare well known in the art.

ELISA and RIA procedures may be conducted such that a TARS standard islabeled (with a radioisotope such as ¹²⁵I or ³⁵S, or an assayableenzyme, such as horseradish peroxidase or alkaline phosphatase), and,together with the unlabelled sample, brought into contact with thecorresponding antibody, whereon a second antibody is used to bind thefirst, and radioactivity or the immobilized enzyme assayed (competitiveassay). Alternatively, TARS in the sample is allowed to react with thecorresponding immobilized antibody, radioisotope- or enzyme-labeledanti-TARS antibody is allowed to react with the system, andradioactivity or the enzyme assayed (ELISA-sandwich assay). Otherconventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. A “one-step” assay may involve contacting antigen withimmobilized antibody and, without washing, contacting the mixture withlabeled antibody. A “two-step” assay may involve washing beforecontacting the mixture with labeled antibody. Other conventional methodsmay also be employed as suitable.

Enzymatic and radiolabeling of a detection agent (e.g., antibodies,binding molecules, etc.) may be carried out by conventional means. Suchmeans will generally include covalent linking of the enzyme to thedetection agent, such as by glutaraldehyde, specifically so as not toadversely affect the activity of the enzyme, by which is meant that theenzyme must still be capable of interacting with its substrate, althoughit is not necessary for all of the enzyme to be active, provided thatenough remains active to permit the assay to be effected. Indeed, sometechniques for binding enzyme are non-specific (such as usingformaldehyde), and will only yield a proportion of active enzyme.

It is usually desirable to immobilize one component of an assay systemon a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed withoutlaborious and time-consuming labor. It is possible for a second phase tobe immobilized away from the first, but one phase may be sufficient.

Enzymes employable for labeling are not particularly limited, but may beselected from the members of the oxidase group, for example. Thesecatalyze production of hydrogen peroxide by reaction with theirsubstrates, and glucose oxidase is often used for its good stability,ease of availability and cheapness, as well as the ready availability ofits substrate (glucose). Activity of the oxidase may be assayed bymeasuring the concentration of hydrogen peroxide formed after reactionof the enzyme-labeled detection agent with the substrate undercontrolled conditions well-known in the art.

Other techniques may be used to detect TARS molecules according to apractitioner's preference based upon the present disclosure. One suchtechnique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci.76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGEgel before being transferred to a solid support, such as anitrocellulose filter. Anti-TARS antibodies (unlabeled) are then broughtinto contact with the support and assayed by a secondary immunologicalreagent, such as labeled protein A or anti-immunoglobulin (suitablelabels including but not limited to ¹²⁵I, horseradish peroxidase andalkaline phosphatase). Chromatographic detection may also be used.

Immunohistochemistry may be used to detect expression of human TARS in abiopsy sample. A suitable antibody is brought into contact with, forexample, a thin layer of cells, washed, and then contacted with asecond, labeled antibody. Labeling may be by fluorescent markers,enzymes, such as peroxidase, avidin, or radiolabelling. The assay may bescored visually, using microscopy, or using any other suitable methods.

TARS Detection Kit

The invention also encompasses kits for detecting the presence of TARSin a biological sample (e.g., a cell sample, tissue sample, tumorsample, etc.). For example, a kit can comprise a labeled or labelableagent capable of detecting TARS polypeptide or nucleic acid (e.g., RNA,DNA, etc.) in a biological sample and a means for determining the amountof TARS in the sample. The agent can be packaged in a suitablecontainer. The kit can further comprise a means for comparing the amountof TARS in the sample with a standard and/or can further compriseinstructions for using the kit to detect TARS nucleic acid orpolypeptide.

This invention in some aspects also provides a kit for measuring humanTARS. Such a kit may include a diagnostic agent (e.g., an antibody orantibody fragments, or binding molecule, etc.) that selectively bindhuman TARS or a set of DNA oligonucleotide primers that allows synthesisof cDNA encoding the polypeptide or a DNA probe that detects expressionof TARS mRNA, etc. In some embodiments of the invention, the primers andprobes may comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ormore nucleotides and hybridize under stringent conditions to a DNAfragment having the nucleotide sequence set forth in GENBANK™ AccessionNo.: NM_152295. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, identity between thesequences.

Methods of Detection

The invention in some aspects provides methods for detecting thepresence of a TARS molecule in a biological sample. The method maycomprise contacting the biological sample with an agent capable ofdetecting TARS polypeptide or nucleic acid molecules (e.g., TARS mRNA orDNA, etc.) such that the presence of TARS is detected in the biologicalsample. An agent for detecting TARS mRNA using methods of the inventionmay be a labeled or labelable nucleic acid probe capable of hybridizingto TARS mRNA. The nucleic acid probe may be, for example, thefull-length TARS cDNA of GENBANK™ Accession No. NM_152295 or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or500 nucleotides in length and sufficient to specifically hybridize understringent conditions to TARS mRNA.

A non-limiting example of an agent that may be used in methods of theinvention for detecting TARS polypeptide is a labeled or labelableantibody capable of binding to TARS polypeptide. Antibodies can bepolyclonal or monoclonal antibodies. An intact antibody, or a fragmentthereof (e.g., Fab or F (ab′) 2) can also be used.

The term “labeled or labelable”, with regard to the probe or antibody,is intended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to thediagnostic molecule (e.g., probe, antibody, binding molecule, etc.), aswell as indirect labeling of the diagnostic molecule by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

Detection methods useful in methods of the invention, including but notlimited to those described above herein, can be used as the basis for amethod of diagnosis of a subject with a tumor and/or cancer (e.g., aprostate tumor, ovarian cancer, etc.), can be used as the basis for amethod of monitoring the progression of the tumor and/or cancer in asubject, or can be used as the basis for a method of determining aprognosis for a subject at risk for developing a cancer or tumor.

In one embodiment, the invention features methods of determining themetastatic potential of a tumor and the methods may involve contacting asample of the tumor (or isolate) with an agent capable of detecting TARSpolypeptide or nucleic acid (e.g., mRNA etc.) such that the presenceand/or level of TARS polypeptide or nucleic acid detected in the tumorsample or isolate, thereby determining the metastatic potential of thetumor. Another aspect of the invention features a prognostic method fordetermining whether a subject is at risk for developing cancer thatinvolves contacting a biological sample obtained from the subject (orisolate of the sample) with an agent capable of detecting TARSpolypeptide or nucleic acid such that the presence and/or level of TARSpolypeptide or nucleic acid is detected in the biological sample orisolate, thereby determining whether the subject is at risk fordeveloping cancer. Yet another aspect of the invention features a methodof diagnosing cancer in a subject and that involves contacting abiological sample obtained from the subject (or isolate of the sample)with an agent capable of detecting TARS polypeptide or nucleic acid suchthat the presence and/or level of TARS polypeptide or nucleic acid isdetected in the biological sample or isolate, thereby diagnosing cancerin the subject.

In some aspects and embodiments of diagnostic methods of the presentinvention may include determining the level of TARS polypeptide ornucleic acid in the sample or isolate. In certain embodiments,diagnostic methods of the present invention may include comparing thelevel of TARs polypeptide or nucleic acid in a sample or isolate withthe level of TARS polypeptide or nucleic acid in a control sample. Inyet another embodiment, a diagnostic or prognostic method of theinvention may also include a step of forming a prognosis or forming adiagnosis, and may also include a step of determining an appropriatetreatment for a subject by a health-care provider based at least in parton the determination of the TARS level in a sample from the subject.

In some embodiments of the invention, a control level of TARS activityis a level determined from cells that do not have the disease orcondition associated with altered TARS activity that is being tested forin the subject's sample. For example, in some embodiments, a controllevel of TARS is a level determined in normal cells that do not have acancer that is suspected to be in the biological sample obtained fromthe subject. In such a case, a primary malignancy of a subject'stumor/cancer cell sample can be diagnosed based on an increase in thelevel of expression of TARS nucleic acid or polypeptide in the subject'ssample as compared to the control that is free of the cancer. In anotherembodiment, the control is from normal cells or a primary tumor and thesubject's tumor sample is a suspected metastatic tumor sample.Acquisition of the metastatic phenotype by the suspected metastatictumor sample can be diagnosed as described elsewhere herein, based on anincrease in the level of TARS polypeptide or nucleic acid in thesubject's tumor sample or bodily fluid compared to the control. Inanother embodiment, determining the level of a TARS molecule can becarried out in conjunction with the determination of one or more otherbiomarkers for cancer or a proliferative disease or condition. Forexample, the level of a TARS molecule (e.g., nucleic acid that encodes aTARS polypeptide, a TARS polypeptide or signature fragment thereof, maybe detected using a panel that also permits detection at least oneadditional molecule whose expression and/or level is may be useful tocharacterize a cancer or other proliferative disease or condition. Forexample, a panel of the invention may include a means to determine thelevel and/or activity TARS and a means to determine the level and/oractivity of each of one or more other markers that may characterize acancer or other proliferative disease or condition to generate anaggregate “biomarker panel signature” to improve diagnosis and treatmentstrategies. Additional biomarkers that may be assessed using anembodiment of a biomarker panel invention include, but are not limitedto: TNF-α, eEF-1α, IL-6, CA125, VEGF, autoantibodies to aminoacyl tRNAsynthetases, and other biomarkers associated with ovarian cancer aspreviously referenced (Altundag et al. 2005; Mor, Visintin et al. 2005).A biomarker panel of the invention may include detection means for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 50, 100or more biomarkers (including each integer in between the listedintegers) in addition to a detection means for TARS.

TARS Nucleic Acid and Polypeptide Sequences and Variations

One aspect of the invention involves isolated nucleic acid moleculesthat encode TARS or biologically active portions thereof, as well asnucleic acid fragments sufficient for use as hybridization probes toidentify TARS-encoding nucleic acid. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA). A nucleic acid molecule maybe single-stranded or double-stranded or may be a double-stranded DNAmolecule. An “isolated” nucleic acid molecule is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, may be free of other cellularmaterial.

In some aspects of the invention, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in GENBANK™Accession No.: NM_152295 The sequence of GENBANK™ Accession No.NM_152295 corresponds to the human TARS cDNA. This cDNA comprisessequences encoding the TARS polypeptide (i.e., “the coding region”, fromnucleotides 1 to 2850), and 3′ untranslated sequences (nucleotides2468-2850). Alternatively, the nucleic acid molecule may comprise onlythe coding region of GENBANK™ Accession No NP_689508 (e.g., nucleotides296-2467).

The invention further encompasses nucleic acid molecules that differfrom the sequence set forth in GENBANK™ Accession No. NM_152295 (andportions thereof) due to degeneracy of the genetic code and thus encodethe same TARS protein as that encoded by the sequence set forth inGENBANK™ Accession No. NM_152295. Accordingly, in another embodiment, anisolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence as set forthin GENBANK™ Accession No. NM_152295. Moreover, the invention encompassesnucleic acid molecules that encode biologically active portions of thesequence set forth in GENBANK™ Accession No. NM_152295.

A nucleic acid molecule having the nucleotide sequence as set forth inGENBANK™ Accession No. NM_152295, or a portion thereof, can be isolatedusing standard molecular biology techniques and the sequence informationprovided herein. For example, a human TARS cDNA library using all orportion of the sequence set forth in GENBANK™ Accession No. NM_152295 asa hybridization probe and standard hybridization techniques (e.g., asdescribed in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd. edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acidmolecule encompassing all or a portion of the sequence set forth asGENBANK™ Accession No.: NM_152295 can be isolated using any suitablemethod, including as a non-limiting example, use of the polymerase chainreaction using oligonucleotide primers designed based upon the sequenceset forth as GENBANK™ Accession No.: NM_152295. For example, TARS mRNAcan be isolated from cells using standard, art-known methods and cDNAcan be prepared using reverse transcriptase and art-known methods.Synthetic oligonucleotide primers for PCR amplification can be designedbased upon the nucleotide sequence set forth in GENBANK™ Accession No.NM_152295 and nucleic acids of the invention can be amplified using cDNAor, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to TARS nucleotide sequencecan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In addition to the human TARS nucleotide sequence set forth as GENBANK™Accession No.: NM_152295, it will be appreciated by those skilled in theart that DNA sequence polymorphisms that lead to changes in the aminoacid sequences of TARS may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the TARS gene may exist amongindividuals within a population due to natural allelic variation. Suchnatural allelic variations can typically result in 1-5% variance in thenucleotide sequence of a gene. Any and all such nucleotide variationsand resulting amino acid polymorphisms in TARS that are the result ofnatural allelic variation and that do not alter the functional activityof TARS are intended to be within the scope of the invention. Moreover,nucleic acid molecules encoding TARS polypeptides from other species,and thus which have a nucleotide sequence that differs from the humansequence set forth as GENBANK™ Accession No.: NM_152295, are intended tobe within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and nonhuman homologues of thehuman TARS cDNA of the invention can be isolated based on their homologyto the human TARS nucleic acid disclosed herein using the human cDNA, ora portion thereof, as a hybridization probe—according to standardhybridization techniques under stringent hybridization conditions, whichare recognized in the art.

In some aspects of the invention, an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequenceset forth in GENBANK™ Accession No.: NM_152295 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein). In one embodiment, the nucleic acid encodesa natural human TARS.

In addition to naturally-occurring allelic variants of the TARS sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes may be introduced by mutation into thenucleotide sequence set forth as GENBANK™ Accession No. NM_152295thereby leading to changes in the amino acid sequence of the encodedTARS protein, without altering the functional ability of the TARSprotein. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues may be made in thesequence set forth as GENBANK™ Accession No. NM_152295. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of TARS polypeptide (e.g., the sequence set forthas GENBANK™ Accession No. NM_152295) without altering the activity ofTARS, whereas an “essential” amino acid residue is required for TARSactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding TARS polypeptides that contain changes in amino acidresidues that are not essential for TARS activity, e.g., residues thatare not conserved or only semi-conserved among members of the subfamily.Such TARS polypeptides differ in amino acid sequence from the sequenceset forth as GENBANK™ Accession No. NM_152295 yet retain TARS activity.In one embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or atleast 99% similar to the amino acid sequence set forth as GENBANK™Accession No.: NM_152295 and retains a level of TARS activity.

To determine the percent similarity of two amino acid sequences (e.g.,GENBANK™ Accession No. NM_152295 and a mutant form thereof), thesequences are aligned for optimal comparison purposes (e.g., gaps may beintroduced in the sequence of one protein for optimal alignment with theother protein). The amino acid residues at corresponding amino acidpositions are then compared. When a position in one sequence (e.g.,GENBANK™ Accession No. NM_152295) is occupied by the same amino acidresidue as the corresponding position in the other sequence (e.g., amutant form of TARS), then the molecules have identity at that position(i.e., as used herein amino acid “similarity” is equivalent to aminoacid “identity”). If two sequences are ‘homologous’ they are descendedfrom a common ancestor. The percent similarity between the two sequencesis a function of the number of identical positions shared by thesequences (i.e., % similarity=number of identical positions/total numberof positions×100). Such an alignment can be performed using any one of anumber of well-known computer algorithms designed and used in the artfor such a purpose.

An isolated nucleic acid molecule encoding a TARS polypeptide that hasidentity to the protein of GENBANK™ Accession No.: NM_152295 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of GENBANK™ Accession No.: NM152295 such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded polypeptide. Mutations can beintroduced into the sequence set forth as GENBANK™ Accession No.NM_152295 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. In some embodiments of the inventionconservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in TARSmay be replaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a TARS coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened forTARS activity to identify mutants that retain TARS activity. Followingmutagenesis of a sequence such as that set forth as GENBANK™ AccessionNo. NM_152295, the encoded protein can be expressed recombinantly andthe TARS activity of the polypeptide can be determined, for exampleusing an assay described herein or other suitable assay.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES Example 1

Threonyl tRNA Synthetase (TARS) is an Angiogenic Chemokine Secreted byEndothelial Cells in Response to VEGF

Materials and Methods for Example 1

Cell Culture, Reagents and Antibodies—

Human umbilical vein endothelial cells (HUVEC) (a gift from C. Holmes,University of Vermont) were grown in Clonetics® EGM®-2 complete media(Lonza, Annandale, N.J.). Borrelidin analog BC194 was a gift from Dr.Barrie Wilkinson, (Biotica). Purified basic-fibroblast growth factor(bFGF) was a gift from J. Spees, Univ. of Vermont. Retinoic acid andcycloheximide were purchased from Sigma-Aldrich, and VEGF and TNF-α werepurchased from Cell Signaling Technology, Danvers, Mass. and Calbiochem,San Diego, Calif., respectively.

Western Blot—

After treatments, cells were harvested into sample buffer containing:0.2 M Tris-HCL, 4% SDS, 4% β-mercaptoethanol, 40% glycerol, 4 μM pyroninY. Extracts were sheared through a 24-gauge syringe. Samples wereseparated by 10% SDS-PAGE and transferred to nitrocellulose membrane andprobed with specific antibody as described (Lounsbury, Beddow et al.1994). Primary antibodies are as follows: Rabbit monoclonal anti-P-eIF2α(1:1000; Cell Signaling Technology, Danvers, Mass.), rabbit monoclonalanti-Cleaved Caspase-3 (1:1000; Cell Signaling Technology, Danvers,Mass.), rabbit polyclonal anti-TARS (1:500; Santa Cruz Biotechnology,Santa Cruz, Calif.). Loading control antibodies were rabbit monoclonalanti-β-actin and anti-β-tubulin (1:1000; Cell Signaling Technology,Danvers, Mass.). Secondary antibodies were HRP-goat-anti-mouse andHRP-goat-anti-rabbit (1:5,000; Jackson Laboratories, Bar Harbor, Me.).

In Vitro Tube Formation Assay—

Tube formation assays were performed as described (Arnaoutova andKleinman 2010; Cassavaugh, Hale et al., 201). Human Umbilical VeinEndothelial Cells (HUVECs) were seeded in 48-well plates (1.5×10⁴cells/well) coated with 100 μl of Matrigel™ Basement Membrane MatrixGrowth Factor Reduced (BD Biosciences, San Jose, Calif.) and incubatedin Clonetics® EGM®-2 complete media (Lonza, Annandale, N.J.) or EGM®-2with reduced serum (0.2% fetal bovine serum). Cells were incubated at37° C. for 6 h then fixed in 10% formalin. Fixed samples were imaged byphase-contrast microscopy or stained with Oregon Green 488 Phalloidin(Molecular Probes, Eugene, Oreg.) then imaged with fluorescencemicroscopy (2× objective). Number of tubes and tube lengths (in pixels)were quantified using the Simple Neurite Tracer (Longair, Baker et al.2011) plug-in on ImageJ software (NIH). Statistical analysis of one-wayANOVA was performed with GraphPad Software. Multiple comparisons wereperformed using the Tukey Test.

Cell Viability—

Cell viability was measured by counting cells in a hemacytometer withTrypan Blue exclusion (Sigma-Aldrich, St. Louis, Mo.) according tomanufacturer's instructions. Measurements were normalized to untreatedcells.

Nascent Protein Synthesis Assay—

Nascent protein synthesis was measured using Invitrogen Click-iT®metabolic labeling reagents [Dieterich, D. C., et al., Nat Protoc 2,532-40 (2007)]. 1). HUVEC cultures were pre-incubated in methionine-freeDulbecco's Modified Eagle Medium (D-MEM) high glucose (Invitrogen, LifeTechnologies, Grand Island, N.Y.) supplemented with 10% dialyzed fetalbovine serum (Invitrogen) containing the control or test compounds.Cycloheximide (50 μM) was used as a positive control. After 45 minutes,25 μM Click-iT® AHA (L-azidohomoalanine) (Invitrogen) was added andcultures were incubated for 3 h. Cells were lysed with 1% SDS in 50 mMTris-HCl with protease and phosphatase inhibitors: 1 mMphenyl-methylsulfonamide, 20 mg/ml aprotinin, and 4 mg/ml leupeptin.Extracts were sonicated and protein concentration was determined byBradford assay. Protein samples were labeled with biotin alkyne (PEG4carboxamide-propargyl biotin) (Invitrogen) using the Click-iT® ProteinReaction Buffer Kit (Invitrogen) according to manufacturer'sinstructions. Equal concentrations of protein were run on a 10% SDS-PAGEand transferred to nitrocellulose membrane, incubated withstreptavidin-HRP reagent (Pierce Thermo Scientific, Rockford, Ill.)followed by reaction with ECL reagent (Pierce) and exposed on film.

Expression and Purification of Human Aminoacyl tRNA Synthetases—

N-terminal His₆-tagged human TARS (ThRS) was expressed and purified fromE. coli Rossetta™ 2(DE3) pLysS competent cells (EMD Millipore,Billerica, Mass.) transformed with derivatives of plasmid pET28ahctThrRS. Transformant cultures were grown in terrific brothsupplemented with 100 mg/ml kanamycin and 100 mg/ml chloramphenicol at37° C. to a cell density of A600=0.6. Expression of TARS was inducedwith 1 mM isopropyl 1-thio-β-D-galactoside overnight at 15° C. Thebacterial pellet was lysed by sonication in buffer A (20 mM potassiumphosphate buffer pH 8.0, 100 mM KCl, 35 mM imidazole, and 5 mMβ-mercaptoethanol) and cleared by centrifugation at 17050×g for 30minutes. Nucleic acids were precipitated by the addition of protaminesulfate to a final concentration of 0.3% followed be centrifugation. Thesupernatant was loaded onto a HisTrap™ FF column (GE Healthcare,Pittsburgh, Pa.) in buffer A and eluted by an imidazole gradient of35-250 mM in buffer A over 20 column volumes. TARS containing fractionswere identified by SDS-PAGE and GelCode™ Blue (Thermo Scientific,Rockford, Ill.), pooled, and dialyzed into buffer B (100 mM potassiumphosphate buffer pH 6.8 and 5 mM (3-mercaptoethanol). The sample wasloaded onto a CHT-Tricom Hydroxyapatite column and eluted over 20 columnvolumes by using a gradient of buffer B to buffer C (500 mM potassiumphosphate pH 8.0 and 5 mM β-mercaptoethanol). TARS-containing fractionswere determined by SDS-PAGE. Buffer B (10 mM HEPES pH 8.0, 100 mM KCl,2.5 mM 3-mercaptoethanol, and 40% glycerol), and stored at −20° C.TARS-containing fractions were pooled and dialyzed into buffer D (10 mMHEPES pH 8.0, 100 mM KCl, 2.5 mM β-mercaptoethanol, and 40% glycerol),and stored at −20° C. Protein concentration was determined by Abs260.The protein purity and stability were evaluated by Coomassie stainfollowing SDS-PAGE, and concentration of active sites was determinedusing a steady state aminoacylation assay (FIG. 5).

The L567V mutant was derived from the wildtype TARS plasmid usingQuikchange II Site-Directed Mutagenesis (Stratagene, Cedar Creek, Tex.[Agilent Technologies Inc., Santa Clara, Calif.]) with the forwardprimer 5′ cac cag tgt gca acc atc cag ctg gat ttc cag gtg ccc atc agattt aat c 3′ (SEQ ID NO:8) and its reverse compliment [5′-gat taa atctga tgg gcc act gga aat cca get gga tgg ttg cac act ggt g-3′ (SEQ IDNO:9)] and transformed into XL1-Blue cells. Colonies positive for themutation were isolated, grown in LB media, and the plasmid purified viaQiagen miniprep kit. The plasmid was then transformed into Rosetta IIcells for use in protein expression using the same protocol as forwildtype TARS.

N-terminal His₆-tagged human leucyl tRNA synthetase (LARS) was expressedusing the plasmid pPROEX hTb-LARS and purified using a similarpurification scheme to the TARS purification with minor modificationsdescribed in (Francklyn, First et al., 2008). The protein purity andstability were confirmed using SDS-PAGE and Coomassie stain (FIG. 9).

Steady State Aminoacylation Assay—

The aminoacylation activities of the TARS constructs were determinedusing modifications to established procedures (Francklyn et al 2008).Briefly, reaction mixtures consisted of 20 mM Tris-HCl pH 8.0, 100 mMKCl, 10 mM MgCl₂, 1 mM dithiothreitol, 2 mM ATP, 2.5 U pyrophosphatase(Roche), 80 μM threonine, 20 μM [¹⁴C]-threonine, and 5 μM of E. coli orhuman tRNAThr. Reactions were initiated with the addition of 0.25-0.75μM TARS and run at 37° C. Aliquots were taken at varying time points andspotted onto Whatmann 3MM paper filters pre-soaked in 5% trichloroaceticacid (TCA). Upon completion, the filters were washed 3 times in excessTCA, once in 95% ethanol, and dried under a heating lamp. The formationof Thr-tRNAThr was detected by scintillation counter and the activitydetermined by linear regression of threonyl-tRNAThr formed per activesite per unit time.

LARS steady state ATPase activity was determined using the sameprocedure as for TARS aminoacylation with the following modifications.The reaction mixture did not include labeled threonine or tRNAThr and 1nM [α-32P] ATP (PerkinElmer, Waltham, Mass.) was added. Reactions wereincubated for 3 minutes at 37° C. and initiated with the addition of 1μM human LARS. At various time points 5 μl aliquots were quenched in 45μl of 500 mM sodium acetate and 0.1% sodium dodecyl sulfate. For eachsample, 1 μl was spotted onto a CCM cellulose PEI F plates (EMD) andresolved via thin-layer chromatography in 0.75 M potassium phosphatebuffer mobile phase. Radioactive signals were detected viaphosphorimaging and AMP production overtime was quantified usingQuantity One v 4.6.6 software (Bio-Rad, Hercules, Calif.).

Chick Chorioallantoic Membrane Assay—

Fertilized chicken eggs (Sunrise Farms, Catskill, N.Y.) day 1-2post-laying were incubated in a humidified incubator at 37° C. for 72 h.Cleaned eggs were cracked and plated in a sterile 10 cm² tissueculture-treated dish and incubated at 37° C. for another 7 days. Ondevelopmental day 10, 1 mm³ sterile gelatin sponge pieces (Surgifoam®;Johnson & Johnson Wound Management, Somerville, N.J.) were placed withinthe outer one-third of the membrane between large vessels. 40 μg/mlhuman bFGF and 2 μg/ml human VEGF were used as pro-angiogenic controlcompounds; 100 g/mL retinoic acid (Sigma-Aldrich, St. Louis, Mo.)diluted in phosphate-buffered saline (PBS) was used as an angiostaticcontrol. All compounds were applied in 10 μl to the CAM every 24 h for72 h. Images were taken using a Leica MZ6 stereomicroscope every 24 h.Compounds were scored according to a modified version of IntensityScoring as previously described (Ribatti, Nico et al. 2006). Briefly,each experimental condition was given a blinded score from 0-5 based onthe change in the extent of vessel convergence and formation inproximity to the sponge from day 0 to day 3. Total score is averagedindividual experimental condition scores from at least 15 replicates.

ELISA and Lactate Dehydrogenase Assays—

Confluent HUVEC cultures (passage 4) were incubated at 37° C. inClonetics® EGM®-2 modified with 0.2% fetal bovine serum with theaddition of 50 ng/ml human VEGF or 50 ng/ml human TNF-α for 6 h asindicated. Culture media supernatants were tested for levels of secretedTARS protein using the Threonyl tRNA Synthetase (TARS) ELISA Kit (USCNLife Science, Wuhan, Hubei, PRC) according to manufacturer'sinstructions. Cell membrane integrity was confirmed using the lactatedehydrogenase assay CytoTox-ONE™ Homogeneous Membrane Integrity Assay(Promega, Madison, Wis.) according to manufacturer's instructions andreported as percent cytotoxicity relative to a lysis control. Levels ofsecreted VEGF were measured using the Human VEGF ELISA kit (ThermoScientific) per manufacturer's instructions.

Quantitative RT-PCR—

Total RNA was extracted from cells using the RNeasy column protocol andcDNA was generated using an Omniscript reverse transcriptase assayaccording to the manufacturer's instructions (Qiagen, Frederick, Md.).Primers and probes for TARS and 32-microglobulin were Assays-on-Demand(Applied Biosystems, [Life Technologies, Carlsbad, Calif.]). RT-qPCR wasperformed using an ABI prism 7700 Sequence Detection System (AppliedBiosystems). The relative quantity of mRNA level was determined usingthe comparative CT (AACT) method using I32-microglobulin to normalizemRNA level (Cassavaugh, Hale et al. 2011).

Endothelial Cell Proliferation Assay—

The MTT-based alamarBlue® (Invitrogen) reagent was used to assess cellproliferation (Ahmed, Gogal et al. 1994). HUVECs were seeded in a96-well dish (1×10³ cells/well) and grown for 48 h in EGM®-2 media.Cells were incubated in 0.2% FBS EGM®-2 media or EGM®-2 complete mediaas indicated; VEGF (50 ng/ml) and media alone served as controls. After48 h, 72 h, or 96 h in culture, 10 μl/well premixed alamarBlue®(Invitrogen) was added and after 3 h at 37° C. the amount of reducedalamarBlue® was quantified by fluorescence (excitation at 530 nm,emission at 590 nm) on a microplate reader (Synergy™ HT, BioTek,Winooski, Vt.).

Transwell Migration Assay—

Migration was assessed using transwell inserts (Svensson, Kucharzewskaet al. 2011). HUVEC cultures were serum-depleted overnight in Clonetics®EGM®-2 modified with 0.2% fetal bovine serum then 5×10⁴ cells wereplated in 90% EBM®-2, 10% EGM®-2 in the upper chamber of 0.2%gelatin-coated 24-well 8 m Transwell® inserts (Corning, Tewksbury,Mass.) with 90% EBM®-2, 10% EGM®-2 media plus 50 ng/ml VEGF, 1-100 nMTARS protein, or 10 nM BC194 in the lower chamber. Cultures wereincubated for 4 h, fixed in 10% formalin, and stained with 10 μg/ml DAPIsolution (Roche) following removal of cells from the top layer of thechamber with a cotton swab. Migrated cells were imaged using a 4×objective on the Olympus IX70 Inverted microscope (Olympus).DAPI-stained nuclei were counted using ImageJ software.

Statistical Analysis—

Data are presented as mean+SEM, and p<0.05 is considered significant.Except where indicated, one-way ANOVA for multiple comparisons wasperformed on all data. A Kruskal-Wallis adjustment was used wherenecessary. All pairwise comparisons were assessed using the Student'st-test.

Results for Example 1 Concentration-Dependent Effects of a TARSInhibitor Reveal a Specific Angiogenic Function for TARS.

Inhibition of TARS by BC194 has been shown previously to reduce in vitroendothelial tube formation (Wilkinson, Gregory et al. 2006); however,because TARS is a component of the protein synthesis machinery, thiseffect could be explained by cell toxicity through the unfolded proteinresponse or apoptosis pathways. By using a range of BC194concentrations, the sensitivity of HUVECs to the anti-angiogenic versuscell stress effects of BC194 was compared. As shown in FIG. 1, thenumber of branches formed by endothelial cells in a tube formation assaywas sensitive to subnanomolar concentrations of BC194, although tubelength was unaffected (FIG. 2). The concentration of BC194 required toaffect tube formation was 100-fold lower than that required to detectthe unfolded protein response (phospho-eIF2α) and apoptosis (cleavedcaspase-3) (FIG. 3). Effects on cell viability, proliferation, andnascent protein synthesis were also unaffected by BC194 atconcentrations below 100 nM (FIG. 4). These data suggest that TARS mayserve a secondary function in angiogenesis signaling that is separatefrom its function in protein synthesis and is highly sensitive toinhibition by BC194. (FIG. 8 provides photomicrographic imagesrepresentative for the data shown in FIG. 4).

Exogenously Added TARS Stimulates Angiogenesis.

In light of the potential role for TARS in angiogenic signaling and thesecreted activity of select other aminoacyl tRNA synthetases (Wakasugiand Schimmel 1999; Greenberg, King et al. 2008), the ability of purifiedTARS to stimulate angiogenesis was tested using the in vitro tubeformation assay. Human His-tagged TARS was expressed in E. coli andpurified by nickel chromatography followed by sequential columnchromatography to produce an active and pure preparation (FIG. 5). Asshown in FIG. 6, addition of TARS to low-serum media significantlyincreased the number of tube branches, suggesting that TARS itself isangiogenic and implicating an extracellular effect for BC194'santi-angiogenic effect on endothelial cells.

To confirm and expand these results, a chorioallantoic membrane (CAM)assay was used to examine a role for TARS in an in vivo angiogenesisenvironment. Daily application of BC194 to a gel sponge on the CAM over4 days inhibited vessel formation at both the basal level and afterstimulation with either bFGF or VEGF (FIG. 7A). Application of TARS tothe CAM stimulated vessel formation and the angiogenic effect wassensitive to BC194, suggesting that the inhibition of angiogenesis byBC194 is not due to off-target effects (FIG. 7B,C). This conclusion wasfurther supported by the finding that a BC194-resistant mutant of TARS,L567V TARS, stimulated vessel formation that was not inhibited byapplication of BC194 (FIG. 7C). Application of Leucyl tRNA synthetase(LARS) to the CAM had no observable effect on vascularization,suggesting that the angiogenic effect is not a property of all tRNAsynthetases (FIG. 9). Together these data support a specific role forextracellular TARS in the activation of the in vivo endothelialangiogenic response.

TARS is Secreted in Response to VEGF and TNF-α.

Although TARS exerts significant pro-angiogenic effects, there was noprior evidence that TARS is physiologically present in the extracellularspace except as a result of cell lysis. To explore the possibility thatTARS is actively secreted, endothelial cells were treated with VEGF orTNF-α followed by measurement of TARS in the media using ELISA. As shownin FIG. 10A, both VEGF and TNF-α stimulated a significant increase inTARS in the media, in an excess of 1000 pg/ml. The TARS present in themedia was not due to cell lysis as confirmed by a cytotoxicity assay(FIG. 10B). The presence of TARS in the media was also not due to anincrease in TARS expression since neither VEGF nor TNF-α induced anincrease in TARS mRNA (FIG. 10D). Furthermore, adding purifiedrecombinant TARS to the cell media did not induce secretion of VEGF asmeasured by ELISA (FIG. 10E). These results support a mechanism wherebyTARS secretion is increased following stimulation of endothelial cellsignaling through VEGF or TNF-α receptors.

TARS Stimulates Endothelial Cell Migration.

An increase in angiogenesis by TARS signaling to endothelial cells couldhave resulted through either an increase in cell proliferation or anincrease in cell migration. Unlike VEGF, TARS did not exert asignificant effect on cell proliferation, and BC194 did notsignificantly reduce the VEGF proliferative response (FIG. 11A).However, TARS significantly increased migration of endothelial cells ina transwell assay to an extent that was similar to VEGF (FIG. 11B). LARSdid not affect migration, indicating that the TARS-mediated effect wasnot a non-selective result of synthetase activity. Importantly, BC194reduced both the migration effects of VEGF and TARS, although the VEGFeffect was less pronounced, suggesting that TARS may play a significantrole in VEGF-mediated endothelial cell migration. This evidence supportsa mechanism for TARS that includes stimulation of endothelial cellmigration that contributes to its angiogenic effect.

TARS may be playing a substantial role in normal and pathogenicangiogenesis as a proangiogenic chemokine activated by endothelial cellsin response to VEGF or TNF-α stimulation (FIG. 12). With this as thefirst report of the novel angiogenic function of TARS, much remains tobe uncovered about how TARS signals to endothelial cells, otherprocesses beyond migration and angiogenesis secreted TARS may beaffecting, and linkages between TARS and tumorigenesis.

Example 2 Database Assessment of TARS in Disease

Database analysis was used to assess TARS expression in cancersincluding Cancer Gene Anatomy Project (CGAP)(Strausberg 2001), GEOdatabase, and Human Protein Atlas (Uhlen, Oksvold et al.). Using theCGAP database, TARS mRNA was found to be over-expressed in cells derivedfrom prostate carcinoma, colon adenocarcinoma, ovarian carcinoma, and incertain stem cell lines. Furthermore, whereas other synthetases werefound to be relatively unchanged, TARS protein was found to beselectively upregulated in ovarian tumors from tissue arrays displayedin the Human Protein Atlas. (www.proteinatlas.org/ENSG00000113407).

A preliminary investigation using GEO data from a prostate cancerprogression study by Tomlins et al. (Tomlins, Mehra et al. 2007)revealed that mRNA levels of TARS exhibited a 2.9 fold increase inprostate carcinoma versus normal (p<0.0001). To expand on thesefindings, GEO dataset GSE6919, 171 sample CEL files (scanned chip imagefiles) were downloaded. GSE6919 is a GEO SuperSeries that includesGSE6604 (normal prostate tissue from 18 patients), GSE6605 (metastaticprostate tumor included 25 samples from 4 patients and 9 sites, somepaired), and paired sets GSE6606 (primary prostate tumor from 65patients), and GSE6608 (normal prostate tissue adjacent to tumor from 63of those patients). Probe-level intensities were background-corrected,normalized, and summarized, and Robust Multichip Average (RMA)statistics are calculated for each probe set and sample as isimplemented in Partek Genomic Suites, version 6.6 Beta (Copyright 2009,Partek Inc., St. Louis, Mo., USA). Sample quality was assessed based onthe 3′:5′ ratio, relative log expression (RLE), and normalized unscaledstandard error (NUSE). Principal Component Analysis (PCA) was also usedto look for outlier samples that would potentially introduce latentvariation into the analysis of differential expression across samplegroups. Based on these analyses, 13 samples were eliminated from furtheranalysis. Additional analysis included assessment of GEO datasets.Results of the analysis, which included a comparison of samples ofnormal and metastatic prostate cancer tumors, indicated that TARS mRNAwas found to be significantly elevated. These findings were selectivefor TARS in that other aminoacyl tRNA synthetases were not elevated inprostate cancer.

Example 3 Analysis of TARS Expression in Prostate Cancer PatientsPatient Selection for IHC Studies—

Using an IRB protocol (CHRMS #:08-218) approved by UVM Committee onHuman Subjects, FAHC patient registries were searched to identifypatients with high grade PCa from 2008-2010 for whom archived tissuesamples were available. The search was confined to those patients forwhom a clinical record was available, and who had all undergoneprostatectomies, and for whom there were clinical samples available. Aninitial set of 54 cases with PC surgeries from 10/08 to 3/10 wascollected in this way. A second group of 79 patients with high-gradedisease was identified with surgeries over the interval 12/99-8/02.

Immunohistochemistry—

The immunohistochemistry procedures were conducted essentially asdescribed (Conant, Penz, et al., 2011). Slide mounted 5 μm tissuesections cut from formalin-fixed, paraffin-embedded (FFPE) prostatecarcinoma specimens were dewaxed by 3×5 mins washes in xylene followedby rehydration through graded ethanol washes (100%, 95%, 70% and 50%;2×3 mins in each). After rinses in Milli-Q ultra-pure water (EMDMillipore, Billerica, Mass.), heat induced epitope retrieval (HIER) wasperformed by immersing the slides in Target Retrieval solution pH 6.0(Dako North America Inc., Carpenteria, Calif.) and heating at 100° C.for 15 mins in a Decloaking Chamber™ Pro pressure cooker (BiocareMedical, Concord, Calif.). Slides were then allowed to cool in thepressure cooker unit for another 20 minutes. After 3×5 minute rinses inTBST (25 mM Tris, 0.15M NaCl, 0.05% Tween 20), slides were immersed in3% H₂O₂/TBST for 15 mins as to inactivate any endogenous peroxidase inthe tissues. After 3×5 min washes in TBST slides were immersed inprotein block, serum-free ((Dako North America Inc., Carpenteria,Calif.) for 15 minutes to block non-specific protein binding sites inthe tissues. Primary antibody (anti-TARS, mouse monoclonal clone 1A9,Abnova, Walnut, Calif.) at a 1:200 dilution was then applied for 30 minat room temperature. As a negative control test, IHC was also performedsubstituting primary antibody with a mouse monoclonal (mAb) IgG1antibody to Aspergillus niger glucose oxidase (Dako North America Inc.,Carpenteria, Calif.). After TBST washes, secondary detection wasperformed by incubating the slides for 30 mins RT with EnVison+Dual Linkpolymer HRP (horseradish peroxidase) reagent (Dako North America Inc.,Carpenteria, Calif.). Following a further series of TBTS washes, slideswere incubated for ˜6 minutes with DAB+chromogen substrate (Dako NorthAmerica Inc., Carpenteria, Calif.) and then rinsed with tap water.Tissues were then counterstained with hematoxylin for ˜7 minutes, rinsedwith TBST and water and the dehydrated through 50%, 70%, 95% and 100%ethanol. Finally, slides were cover-slipped with Cytoseal mountant(ThermoFisher Scientific, Waltham, Mass.) for viewing by bright-fieldmicroscopy.

Imaging Details—

Images of IHC were captured at the Micrscopy Imaging Center at the UVMCollege of Medicine using an Olympus BX50 light microscope, QImagingRetiga 2000R camera, and QCapture Pro Software. The final images usedfor standards in grading are seen in FIG. 13A.

Immunohistochemistry Scoring—

The stained slides were initially scored by a primary pathologist, andthen a secondary review was provided two other expert pathologists. Inthe scoring procedure, the pathological grade and the intensity of TARSimmunochemical staining were evaluated independently. In thepathological grading, each slide was evaluated by assigning differentregion of the slide to benign (non-tumor) and tumor. The total tumorarea was further subdivided into Gleason primary pattern 3, 4, or 5, anda % of tumor region estimated for each. (There are no Gleason scoresbelow 2, because such patients were never subjected to prostatectomies.)The AJCC criteria were used to make these pathological assessments. Thepathological assessment also included the recording and grading of HGPIN(a precursor lesion to PCa) and several benign controls, including BPHand atrophy (characterized by small, hyperchromatic nuclei with noprominent nucleoli). The experimental analysis noted tissue stainingpattern (diffuse, focal, or scattered), and any additional notes (suchas tertiary Gleason grade, lymph node metastasis, or extraprostaticextension (EPE). In cases where there were uncertainties and/orambiguities, the original H&E stained slides corresponding to each casewere referenced. The TARS IHC staining intensity was gradedindependently, using a semiqualitative scale from 0-3 (0=negative,1=mild, 2=moderate, and 3=strong). A set of reference slides that servedto calibrate the scoring procedure is shown in FIG. 13A. The regions ofeach slide corresponding to the various Gleason scores were eachindependently scored for TARS, as was the “benign” region.

Statistical Analysis.

Univariate statistics were used to determine the significance betweenTARS staining and tumor type. Secondary t-tests were done to correlateTARS intensity with progression from Gleason 3 to 4 and to correlateTARS intensity with PSA and biochemical failure.

ELISA Assays.

TARS ELISA was perform on neat serum samples, according themanufacturer's (CUSABIO Biotech, Wuhan, P.R. China) instructions. Theserum samples from four age matched male non-cancer subjects was used asthe control group.

Results for Example 3 Immunohistochemical Analysis of TARS Expression inProstatectomy Sections.

To assess the relationship between TARS expression and prostate cancerprogression, patient tumor samples were analyzed by immunohistochemistryand scored by intensity as shown in FIG. 13A. Statistical analysis ofthe data concluded that TARS protein levels are increased in tumors withGleason score of 3 and above (FIG. 13B). In addition, a post-analysis ofthe TARS intensity found a significant increase in expression duringprogression from Gleason score 3-4 with a mean difference of 0.304, anda p-value of 0.0001.

TARS expression was also compared with 10-year outcome. When TARSstaining of the various anatomical grades was examined, there was astrong relationship between Gleason 5 staining and elevated PSA at10-years. Specifically, a one unit increase in TARS staining on theGleason 5 portion of the slide increases the odds by a factor of 2.211that subject will experience biochemical failure. Taken together theseresults suggest that TARS expression correlates with diagnosis ofprostate cancer, progression of disease and likelihood of biochemicalfailure.

Analysis of Circulating TARS Levels in Prostate Cancer Patients.

The essential features of a useful human biomarker are that it bepresent in medium that can be readily obtainable in a non-invasivefashion (e.g. serum or urine), that it be readily quantifiable using arobust and repeatable assay, and that it provide useful information thatreflects on subject disease state. As part of the very initial processof TARS biomarker discovery, serum samples were collected from 10consenting subjects of the Fletcher Allan Urology Clinic. This small setincluded patients at various points along the prostate cancerdiagnosis/treatment continuum, including immediately after diagnosisprior to treatment; under active surveillance; and under androgendeprivation therapy following prior radiation or prostatectomy surgery.Serum samples from four age and gender matched control subjects werealso analyzed. All samples were measured in duplicate, and the valuesreported in FIG. 13C are mean values.

The mean value of the control samples was 105±19.5 pg/mL. Two patients(TARS 0012 and TARS 0013) had values higher than the controls, and theother eight patients all exhibited values lower than the controls. Inthree cases (TARS 0014, TARS 0016, and TARS0018) the levels ofcirculating TARS were undetectable, and significantly decreased levelswere seen in three others (TARS0011, TARS0014, and TARS 0017). In threeof the six cases where TARS levels were significantly decreased or notdetectable, the patients were on androgen deprivation therapy. In onepatient under androgen deprivation therapy, TARS levels were increased50% relative to the controls. Notably, the two patients with TARS levelsclosest to the controls had either received no treatment or were underactive surveillance. These data allow several important conclusionsregarding the potential utility of TARS as a prostate cancer biomarkerto be drawn. First, the TARS enzyme can be readily detectable in humanserum samples by a conventional and commercially ELISA kit without anyextensive modification or adaptation. Secondly, the variation in levelsamong different subjects is within the dynamic detection range of thekit. Thirdly, the values seen in untreated or active surveillancepatients were closer to the values seen in the controls than samplesderived from patients who had undergone past surgery/radiationtreatments and were currently under androgen deprivation therapy. Thisprovides initial support for the hypothesis that circulating TARS levelschange in prostate cancer patients in response to treatment. It isnoteworthy that the significant drop in TARS levels seen with patientsunder androgen deprivation suggests that TARS expression is at leastpartially under the control by the androgen receptor.

Example 4

TARS Interacts with VHL and its Inhibition Interferes with the OvarianCancer Cell Response to Hypoxia.

Materials and Methods for Example 4 Co-Immunoprecipitation—

Plasmids expressing biotinylatable TARS (pTARS) and myc-tagged VHL(pVHL) were transfected into HEK293 cells, and then extracts wereprepared. Biotin-TARS was precipitated using streptavidin-coupled beads.Myc-VHL was precipitated using anti-myc antibodies. Precipitates wereseparated by SDS-PAGE, transferred and blots probed with anti-TARSantibody or anti-myc (VHL) antibody.

Western Blot—

After treatments, cells were harvested into sample buffer containing:0.2 M Tris-HCL, 4% SDS, 4% β-mercaptoethanol, 40% glycerol, 4 μM pyroninY. Extracts were sheared through a 24-gauge syringe. Samples wereseparated by 10% SDS-PAGE and transferred to nitrocellulose membrane andprobed with rabbit polyclonal anti-TARS (1:500; Santa CruzBiotechnology, Dallas, Tex.) or monoclonal anti-HIF-1α (BD TransductionLaboratories, [BD Biosciences, San Jose, Calif.]) (Lounsbury, Beddow etal., 1994). Secondary antibodies were HRP-goat-anti-mouse andHRP-goat-anti-rabbit (1:5,000; Jackson Laboratories, Bar Harbor, Me.).

Mass Spectrometry.

Culture dishes were seeded with 2×10⁶ human embryonic kidney cells(HEK293) and maintained in DMEM (Mediatech, Manassas, Va.) supplementedwith 10% fetal bovine serum (Gibco, Carlsbad, Calif.),penicillin/streptomycin (Gibco), and L-glutamine (Gibco) at 37° C. and5% CO₂ in a humidified incubator. Cells were transfected bypolyethylenimine with plasmids encoding a TARS construct with C-terminalHA tag and BirA biotinylation site, BirA, and C-terminally myc-taggedVHL. Control experiments substituted an empty vector plasmid for theTARS construct. Following a 48 hour incubation, cells were lysed with 1%Triton X, 0.5% NP-40, 140 mM NaCl, 25 mM Tris-HCl pH 7.6, and 1 CompleteMini protease inhibitor tablet (Roche) per 10 ml. TARS was then“pulled-down”with streptavidin immobilized on magnetic beads(Invitrogen, Dynabeads MyOne Streptavidin) and unbound proteins werewashed away with three exchanges of lysis buffer. The bound proteinswere eluted from the beads through boiling and resolved on a reducing,SDS-PAGE gel. Major bands and their empty vector counterparts weredetected using SilverSNAP Stain Kit II (Pierce) and excised. Fragmentswere typically digested using the in gel procedure for ProteaseMAXSurfactant (Promega, Madison, Wis.) according to the manufacturersspecifications. Briefly, free cysteines were alkylated by incubationwith 55 mM iodoacetamide:50 mM NH₄HCO₃ followed by trypsin digestion (2ng/μl) in 0.01% ProteaseMAX surfactant:50 mM NH₄HCO₃. Peptides wereanalyzed by electospray ionization (ESI) liquid chromatography massspectrometry (LC-MS). Samples were resolved over a fused-silicamicrocapillary MagicC18 LC column (12 cm×100 μm i.d.) using a 5-50%acetonitrile gradient in 0.1% formic acid. Spectra were obtained usingcollision-induced dissociation with an LTQ linear quadrupole iontrap-Orbitrap mass spectrometer (Thermo Electro, San Jose, Calif.) andanalyzed using SEQUEST (Bioworks software package, version 3.3.1; ThermoElectron, San Jose, Calif.). Acquired TARS data were compared to emptyvector equivalents in order to identify non-specific interactions.

Results for Example 4

An interaction between TARS and VHL may affect hypoxia signaling. VHL isthe E3 ubiquitin ligase for Hypoxia inducible factor-1α (HIF-1α), thusif TARS interferes with VHL activity, it may influence the induction ofHIF-1α by hypoxia. Shown in FIG. 14, the TARS inhibitors BC144 and BC194diminished the levels of HIF-1α protein stabilization in SKOV3 ovariancancer cells responding to hypoxia. The effect was through stabilizationas there was no change in HIF-1α transcription. Accordingly, it washypothesized that the lowering of HIF-1α levels by BC194 occurs as aconsequence of a TARS' interaction with VHL.

A large-scale study examining protein-protein interactions in humancells featured the immunoprecipitation of flag tagged bait proteins,followed by the LC-ESI/MS analysis of interacting proteins. Using theVon Hippel Lindau tumor suppressor as bait, TARS was identified as apotential binding partner. This result was confirmed in two independentapproaches. In the first of these experiments, HEK cells weretransfected with expression plasmids for TARS-[hemagglutinintag]—[biotinylation recognition] and VHL-[myc-tag]. The TARS constructpossessed an appended peptide tail that served as recognition site forthe E. coli biotin ligase, whose gene was also transfected into cells.As shown in FIG. 15A, when biotinylated TARS is precipitated incubationof the extracts with streptavidin beads, the VHL protein isco-precipitated. Conversely, immunoprecipated VHL will alsoco-precipitate full-length TARS. The region of TARS that interacts withVHL was explored by a comparison of the structure of theVHL-ElonginB-ElonginC complex to TARS. ElonginB which is a component ofthe complex, can be readily superimposed with the N-terminal domain ofTARS (106 residues aligned; 2.71 r.m.s.d.; p value=0.0019). To confirmthe significance of this structural relationship, HEK cells weretransfected with plasmids expressing the N-terminal domain of TARS−[hemagglutinin tag]-[biotinylation recognition] and VHL-[myc-tag], andthen TARS N1 domain was precipitated with streptavidin beads. Thisanalysis showed that the N1 domain precipitated VHL more efficientlythan the full length TARS (FIG. 15B). Hence, the N1 domain is likely tobe one of the major interaction domains with VHL.

In order to provide additional validation of the proposed VHL-TARSinteraction, and perform the converse experiment of the original Ewinget al experiment (Ewing, Chu, et al. 2007), the proteins that associatewith TARS in vivo were identified by precipitating biotinylated TARS,resolving all proteins by SDS polyacrylamide gel electrophoresis, andthen subjecting isolated bands to mass spectrometry analysis. Theresulting TARS binding partners that were identified are shown in FIG.16. As a control, a parallel lane was run with proteins precipitatedfrom HEK cells transfected with an “empty” plasmid that does not overproduce TARS, or any other protein that can be biotinylated. Allpeptides that were common to both the TARS plus and control weresubtracted from the final results. The “TARS plus” experiments wereperformed in the presence and absence of plasmids expressing VHL. Theseexperiments identified a number of partners for TARS, including VHL, theglutamyl-prolyl tRNA synthetase (EPRS), poly[ADP-ribose] polymerase 1(PARP), and elongation factor 1 alpha 1 (eEF1A1). Several other proteinswere also detected as single peptides.

Example 5 Angiogenesis Related Secondary Functions of HumanThreonyl-tRNA Synthetase Materials and Methods for Example 5 TARSPreparation and Nucleotide Assays.

TARS purification and active site determination were performed aspreviously described (Williams, Mirando et al., 2013). TARSnon-canonical catalytic activities were characterized usingmodifications from published methods (Guo, Chong et al. 2009). For Ap4Areactions 5 μM of wildtype TARS or R442A TARS were incubated for 10minutes on ice with 2 mM threonine, and 10 μM BC194 or borrelidin asindicated in FIG. 17C. Reactions were initiated using 2 mM ATP withtrace amounts of [α-³²P]-ATP as label. At specific time points aliquotswere quenched in 3 volumes of 0.1% SDS, 400 mM sodium acetate andresolved on polyethyleneimine-cellulose plates by thin-layerchromatography (TLC) in 3 M NH₄ (SO₄)2 and 2% EDTA. Radioactive countswere identified by phosphorimaging and products quantified as fractionsof total ATP added. The quantitation of product was normalized to takeinto account the fact that each Ap4A molecule has two equivalents ofradioactive phosphorus.

Ap4G and GTPase assays were performed as above with the followingexceptions: Ap4G reactions always included 2 mM ATP and 2 mM threonineand 10 μM BC194 where indicated in FIG. 17D. For GTPase assays, ATP andthreonine were not present in all reactions but were included incombination with, 10 μM tRNA^(Thr), 10 μM BC194, and 10 μM borrelidinaccording to FIG. 17F. Adenylation conditions consisted of ATP andthreonine and aminoacylation conditions further included tRNA^(Thr).Reactions were initiated using 2 mM GTP with trace amounts of[α-³²P]-GTP. The components were resolved by TLC using the mobile phases750 mM KH₂PO₄, pH 3.5 for GTPase data and 3 M NH₄ (SO₄)₂ and 2% EDTA forAp4G. Unlike with Ap4A, Ap4G involves only the incorporation of onelabeled nucleotide and does not require the 0.5 correction factor.

CAM assays.

Fertilized chicken embryos were cultured ex-ova and, starting atdevelopmental day 10, agents were applied daily to gelfoam sponges onthe CAM. Images were recorded daily over 72 h and scored blindlyaccording to a modified version Intensity Scoring as previouslydescribed (Ribatti, Nico, 2006). Additional details regarding methodsare provided in the Methods and Materials section of Example 1.

Results for Example 5

An important scientific question is the extent to which thepro-angiogenic functions of TARS are directly linked to aminoacylationfunction. Alternatively, stimulation of angiogenesis might be linked toalternate catalytic functions, employing substrates and products thatare distinct from those aminoacylation. To directly test whetheraminoacylation is required for stimulation of angiogenesis, a mutantversion of hTARS was produced in which an essential arginine in theactive site (Arg442) was substituted with alanine. As shown in FIG. 17A,the resulting mutant protein displayed essentially no aminoacylationactivity. Next, the chorioallantoic membrane (CAM) assay was used toinvestigate whether loss of aminoacylation was associated with loss ofangiogenesis stimulating activity. As shown in FIG. 17B, R442A TARSdemonstrated angiogenesis stimulating abilities that were virtuallyindistinguishable from wild type. Notably, the uninhibited version ofR442A TARS had an average CAM score that was equal to that of wild type,and R442A displayed a similar level of inhibition of angiogenesisstimulus in the presence of BC194. On the basis of these experiments, itwas concluded that aminoacylation function is not required for thestimulation of angiogenesis.

In light of the previous observation that aminoacylation activity is notrequired to stimulate angiogenesis, the possibility that an alternativecatalytic function might be involved was explored. One such alternativeis the production of diadenosine tetraphosphate (Ap4A), which isproduced by human lysyl-tRNA synthetase in immune cells that becomeactivated by antigen. The Ap4A produced by LysRS binds to Hint,liberating the associated microphthalmia transcription factor (MITF) toexecute a complicated program of gene expression (Lee, Nechushtan et al,2004; Ofir-Birin, Fang et al. 2013). This confirms the role of Ap4A asan intracellular signaling molecule. There is also data to suggest thatAp4A can function as an extracellular signaling molecule (McLennan 2000;Delicado, Miras-Portugal et al., 2006). Significantly, Ap4A is releasedextracellularly from platelets, and is capable interacting with the P2Yand P2X receptors. These interactions have the potential of modulatingangiogenesis in endothelial cells (Chang, Yanachkov et al. 2010;Roedersheimer, Nijmeh et al., 2011).

As shown in FIG. 17C, significant production of Ap4A was observed forwildtype TARS (0.1391 Ap4A/active site/min) and E. coli ThrRS (0.5612Ap4A/active site/min; data not shown). In contrast, no appreciableactivity was observed for the aminoacylation-deficient, R442A TARS or inabsence of threonine, suggesting that the adenylate intermediate isessential for dinucleotide formation. Treatment with BC194 andborrelidin resulted in a 31.0% and 59.4% decrease in activityrespectively. This reduction in activity is not unsurprising as previousreports indicate that borrelidin compounds inhibit aminoacylation at thelevel of adenylate formation (Ruan, Bovee et al., 2005). However, the 10μM concentration used for both compounds is in great excess of thecalculated K_(i) values (4.1 nM for BC194; see Williams, Mirando et al.2013) and 4.6 nM for borrelidin, data not shown) suggesting that maximuminhibition still allows for reduced formation of the adenylateintermediate. A possible explanation for this modest drop in activitycompared to the nearly complete inhibition of aminoacylation at similarconcentrations is that borrelidin compounds block tRNA^(Thr) binding aswell; encouraging the small amount of adenylate that does form to beused in the synthesis of dinucleotide compounds. Similar results wereobserved (FIG. 17D) in studies of Ap4G: rates for wildtype TARS and E.coli ThrRS (data not shown) were comparable to Ap4A data (0.1639 and0.5612 Ap4G/active site/min respectively). The reaction was notcatalyzed by R442A TARS and required the presence of both ATP andthreonine, suggesting that adenylate formation was still a requirement.Since threonine alone was not sufficient to form a significantdinucleotide product, Gp4G formation is unlikely. Once again, treatmentwith BC194 reduced the reaction rate by 57%. Given the similaritiesbetween the two processes, it is likely that Ap4A and Ap4G formationoccurs at the same site in the enzyme; however, the exact residuesinvolved remains to be determined.

Another reaction in which aminoacyl-tRNA synthetases can potentiallymodulate signaling is GTP hydrolysis. Recent published work thatindicates that LeuRS may contribute amino acid sensing properties to theMammalian Target of Rapamycin complex (mTOR) by virtue of interactionswith the Rag GTPase a mediator of amino acid signaling to mTORC1(Bonfils, Jaquenoud et al., 2012; Han, Jeong et al., 2012). In contrastto the dinucleotide synthesis, TARS GTPase activity differs greatly inresponse to similar treatments. As shown in FIGS. 17E and 17F, a directstimulation of GTP hydrolysis by TARS was observed in both the wildtypeand R442A TARS (0.3033 and 0.2137 GDP/active site/min respectively) butnot in E. coli ThrRS. Furthermore, there was no observable change upontreatment with either BC194 or borrelidin. Taken together, these datawould suggest that TARS GTPase activity is specific to the human enzyme(relative to E. coli) and does not require the same catalytic residuesas aminoacylation. Despite this apparent disconnect to aminoacylation,GTPase activity is responsive to the availability of canonicalsubstrates. While threonine appears slightly stimulating (26% increasein activity) ATP, adenylation, and aminoacylation conditions decreaseactivity by 77%, 90%, and 89% respectively. Given that all of theseconditions require ATP, it may be that the two nucleotides compete forthe same site. However, there is not enough information to rule out anallosteric form of inhibition as well. Interestingly, the formation ofAp4G requires ATP to be present but maintains the same activity of Ap4A,suggesting that the use of GTP in the synthesis of dinucleotides is notsimilarly regulated.

Example 6 TARS is Overexpressed in Ovarian Cancer Materials and Methodsfor Example 6

Ovarian Cancer Study Group—

The ovarian cancer studies were approved by the University of Vermont'sinstitutional review board (CHRMS 01-026 and M12-004). The study groupconsisted of 58 patients diagnosed with epithelial ovarian cancer atFletcher Allen Health Care/University of Vermont between 1999 and 2001.The control group consisted of 16 women who underwent oophorectomies forgynecologic reasons (other than ovarian cancer) and the final pathologydemonstrated normal ovarian tissue. Serum and paraffin embedded samplesfrom both the study and control group were obtained after adequateportions of the samples were evaluated for pathologic diagnosis.Histological subtype was based according to the WHO criteria and gradingof tumors. Formalin-fixed, paraffin embedded tissue samples from eachpatient were retrieved. Three serial sections (6 μm) from each specimenwere cut and transferred to slides, then analyzed usingimmunohistochemistry to measure the expression of VEGF and TARS aspreviously described (Wong, Wellman et al., 2003). Afterdeparaffinization and rehydration, slides were incubated at 97° C. for15 min with DAKO target retrieval solution, containing 100 mM Tris base,pH 10.5 and 0.1% Triton X-100. Immunoperoxidase staining was performedusing the mouse ImmunoCruz staining system (Santa Cruz Biotechnology,Santa Cruz, Calif.) according to the manufacturer's protocol. Antibodieswere mouse monoclonal anti-VEGF (1:100, Santa Cruz Biotechnology) andmouse monoclonal anti-TARS (1:100, Clone 1A9 Abnova, Taipei City,Taiwan). Normal mouse IgG was used as a negative control. Afterimmunoperoxidase staining, cells were lightly stained with Mayers'hematoxylin and eosin. Slides were dehydrated through xylenes thenmounted with coverslips using Cytoseal 60 (Richard-Allan Scientific,Kalamazoo, Mich.). Images were obtained using an Olympus BX50 lightmicroscope coupled to a CCD camera and Metamorph image capture software.Slides were scored for the expression of VEGF and TARS on a scale of 1-4where 1=no staining and 4=intense staining. TARS ELISA was performed onundiluted serum following the manufacturer's instructions (CusabioBiotech; Wuhan, P.R.China). Statistical significance was determinedusing Kruskal-Wallis test. Correlation between TARS and VEGF expressionwas evaluated using multiple regression correlation coefficient.

Results for Example 6

It has previously been shown that VEGF is overexpressed in ovariancancer and correlates with progression of disease (Wong, C. et al.(2003) Gynecol Oncol 91 (3), 513-517). To determine whether TARS isdysregulated in human ovarian cancer, immunohistochemical staining forTARS was performed on patient tumor sections and correlated withstaining of VEGF and serum levels of TARS. In the samples analyzed, TARSstaining colocalized with VEGF and was selectively overexpressed in thetumor cells (FIG. 18). In addition, TARS serum levels significantlycorrelated with TARS tissue levels, supporting further analysis of TARSas an indicator of ovarian cancer (FIG. 18). Scoring and statisticalanalyses of data from all of the patients is ongoing and additionalpatients will be recruited to determine if TARS levels correlate withstage of disease as well as patient outcome leading to Example 7.

Example 7 Identifying a Clinical Relationship Between Cancer and TARSProtein Level and Activity in Tissue and Serum

Studies are performed establishing a clinical connection by correlatingexisting measurements of overexpressed HIF-1α and VEGF in human ovariancancer (Wong, Wellman et al., 2003) with levels of TARS protein intissue and serum. For these experiments immunohistochemistry is used inovarian tissue sections and ELISA of blood samples from ovarian cancerpatients to correlate TARS levels with angiogenesis and outcome in humanovarian cancer. TARS levels are elevated in ovarian cancer patients in asimilar pattern as VEGF expression and TARS is expected to be detectedat higher levels in the serum of patients with TARS-positive ovariancancer.

Results of these experiments better define the newly discovered pathwaywhereby cells regulate angiogenesis through unconventional signaling byan aminoacyl tRNA synthetase. These experiments also determine theanti-angiogenic activity of TARS inhibitors, which suggest theirdevelopment for and use as a therapeutic in ovarian and otherangiogenesis-dependent cancers. Furthermore, these experiments determineif TARS secretion can be used as a means of detecting angiogenic ovariancancer to assist in earlier diagnosis and improved treatment regimensfor ovarian cancer patients.

Studies are performed that include measuring TARS expression in tumorsand serum obtained from cancer patients, including but not limited toovarian cancer patients, prostate cancer patients, etc. In the studies,angiogenesis markers such as PECAM are compared with control levels andthe cancer's status, stage, and progression and the subject's prognosisis determined. Additional types of cancers tested are metastaticcarcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma;prostate cancer; androgen independent prostate cancer; Kaposi's sarcoma;colorectal cancer, hepatobilliary cancer, gastric cancer, epithelialovarian cancer; lung cancer, and/or mesothelioma.

Experiments are performed and TARS activity and/or expression ismeasured in samples from one or more subjects that have or are suspectedof being at risk of having additional diseases and conditions such asangiogenesis-associated disorder in which the level of TARS is increasedas compared to a normal control level. A TARS level is determined toassess a cancer, a tumor, a hemangioma, a vascular overgrowth, a venousmalformation, an arterial malformation, overweight, maculardegeneration, an inflammatory disease, psoriasis, diabetes, orrheumatoid arthritis.

Additional studies are performed and TARS activity and/or expression isdetermined in samples from one or more subjects that have or aresuspected of being at risk of having additional diseases and conditionssuch as angiogenesis-associated disorder in which the level of TARS isdecreased as compared to a normal control level. A TARS level isdetermined for a tissue implant, an organ implant, ischemia, cardiacinfarction, tissue trauma, cartilage to bone transformation, stroke,surgery, pregnancy, macular degeneration, and/or vascular occlusion.

The stage and prognosis for one or more of the diseases and conditionslisted above elsewhere herein are assessed by determining the level ofTARS in a sample and comparing the level to a level in a control sample.

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Additional references are cited in Specification and Examples.

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Although several embodiments of the present invention have beendescribed and illustrated herein, those of ordinary skill in the artwill readily envision a variety of other means and/or structures forperforming the functions and/or obtaining the results and/or one or moreof the advantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto; the invention may be practiced otherwise than asspecifically described and claimed. The present invention is directed toeach individual feature, system, article, material, and/or methoddescribed herein. In addition, any combination of two or more suchfeatures, systems, articles, materials, and/or methods, if suchfeatures, systems, articles, materials, and/or methods are not mutuallyinconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified, unless clearly indicated to the contrary.

All references, patents and patent applications and publications thatare cited or referred to in this application are incorporated in theirentirety herein by reference.

What is claimed is:
 1. A method of diagnosing a subject as having, or atincreased risk of developing, a disorder associated with increasedthreonyl-tRNA synthetase (TARS) activity, the method comprising (a)obtaining a biological sample from a subject; (b) measuring the amountof a TARS molecule in the biological sample; and (c) comparing the levelof the TARS molecule in the biological sample to a control level of theTARS molecule, wherein a significant increase in the level of the TARSmolecule in the subject biological sample compared to the control leveldetermines that the subject has or is at increased risk of developingthe disorder associated with increased TARS activity.
 2. (canceled) 3.The method of claim 1, wherein the control is one or more of a level ofthe TARS molecule characteristic of biological samples from individualsfree of the disorder associated with increased TARS activity, and alevel of the TARS molecule in a sample obtained from the subject at adifferent time than the sample obtained in step (a).
 4. The method ofclaim 1 wherein the level of the TARS molecule is measured by measuringa secreted TARS molecule.
 5. The method of claim 1, wherein the level ofthe TARS molecule is measured by measuring a non-secreted TARS molecule.6. The method of claim 1, wherein the level of the TARS molecule ismeasured by measuring the activity of the TARS molecule.
 7. The methodof claim 1, wherein the disorder associated with increased TARS activityis an angiogenesis-associated disorder.
 8. The method of claim 7,wherein the angiogenesis-associated disorder is a cancer, a tumor, ahemangioma, vascular overgrowth, venous malformation, arterialmalformation, overweight, macular degeneration, inflammatory disease,psoriasis, diabetes, or rheumatoid arthritis.
 9. The method of claim 8,wherein angiogenesis-associated disorder is a cancer.
 10. The method ofclaim 9, wherein the cancer is a metastatic cancer.
 11. The method ofclaim 1, wherein the biological sample is a sample of blood, tissue,serum, urine, stool, sputum, cerebrospinal fluid, or supernatant fromcell lysate.
 12. The method of claim 1, wherein the biological samplecomprises a cell or tissue.
 13. The method of claim 1, wherein the TARSmolecule comprises a TARS polypeptide.
 14. The method of claim 1,wherein the TARS molecule comprises a TARS-encoding nucleic acid. 15.The method of claim 1, wherein a means of the measuring comprises animmunological assay, nucleic acid determination, mass spectrometry, TARSaminoacylation assay, TARS active site determination assay, or a TARSbinding assay comprising a TARS-binding reporter molecule.
 16. Themethod of claim 15, wherein the immunological assay comprises an ELISAassay.
 17. The method of claim 1, further comprising selecting atreatment regimen for the subject based at least in part on thecomparison of the level of the TARS molecule in the biological sample tothe normal control level of the TARS molecule.
 18. The method of claim17, wherein the treatment regimen comprises administering one or more ofa medicament, surgery, radiation, or chemotherapy to the subject. 19.The method of claim 17, wherein the treatment regimen comprises stoppinga prior administration regimen of one or more of a medicament, surgery,radiation, or chemotherapy to the subject.
 20. (canceled)
 21. The methodof claim 1, further comprising administering one or more of amedicament, surgery, radiation, or chemotherapy to the subject to treatthe disorder, wherein the selection of the one of more of themedicament, surgery, radiation, or chemotherapy is selected based, atleast in part, on the diagnosis. 22-53. (canceled)
 54. The method ofclaim 1, wherein the method also includes measuring the amount of onemore additional biomarker molecules in the biological sample; and (d)comparing the level of the one or more measured biomarker molecules tocontrol levels of each of the one or more biomarker molecules, wherein asignificant change in the level of one or more of the biomarkermolecules in the subject biological sample compared to the control levelis an indication that the subject has or is at increased risk ofdeveloping the disorder associated with increased TARS activity.